Background: C. difficile is one of the primary infectious causes of morbidity and mortality in Canada. Colonized patients can pose a risk to others as a factor in the transmission and development of hospital-associated C. difficile infections. Despite immense efforts and resources invested in the reduction in C. difficile transmission within Canada and Hamilton Health Sciences – further reduction in these rates are unlikely, and novel screening strategies are imperative in this field of study. Methods: This project was a retrospective cohort study of adult in-patients admitted to either The Juravinski, Hamilton General, or St. Joseph’s Healthcare Hamilton Hospitals from January to April 2018 and September 2018 to August 2019. MSRA/VRE swabs were collected during admission or through universal point prevalence screening and subsequently tested for colonization. Results: From the 1056 patients in the data sample, 72 were colonized with asymptomatic C. difficile resulting in a prevalence rate of 6.81%. In-patient point prevalence screening strategies identified more carriers than admission swabs alone (p < 0.001). Risk factors for colonization on admission were being female (OR 2.66, 95% CI 1.02-8.33) and previous CDI (OR 4.76, 95% CI 1.49 – 13.86). During hospitalization, risk factors for colonization were previous CDI (OR 4.75 95% CI 2.14-9.94) and recent hospitalization within the last 12 months (OR 2.35, 95% CI 1.30-4.42). The multi-level Cox PH model identified those with a recent hospitalization (OR 2.21, 95% CI 1.32 – 3.73) and those with previous CDI (OR 2.40, 1.34 – 4.30) were twice as likely to develop asymptomatic C. difficile colonization throughout hospitalization. Conclusion: The addition of universal point prevalence screening in addition to admission screening helped identify more than double the amount of carriers in the population. Moreover, a previous hospitalization, previous CDI, and being female may indicate patients at the highest risk of colonization. / Thesis / Master of Science (MSc) / C. difficile infection (CDI) is a severe infectious disease. Patients with asymptomatic C. difficile present a risk to others as they can contribute to the spread and development of hospital-associated CDI. We are currently unsure of the proportion of adult in-patients colonized with asymptomatic C. difficile. Identifying these carriers early on in their hospital stay is imperative to reduce CDI rates in health care settings. Our study objectives were to determine the best screening strategies to identify asymptomatic carriers, identify risk factors for carriage, and understand the transition from asymptomatic C. difficile to symptomatic CDI. We demonstrated that being female, being recently hospitalized or previously having CDI may increase a patient's risk of being an asymptomatic carrier. Also, timely screening throughout a hospital stay in addition to admission screening helped identify more colonized in-patients. Lastly, we determined that 1 in 5 carriers would go on to develop symptomatic CDI infection.

Healthcare-acquired infections (HAIs) cost the National Health Service (NHS) in England in excess of £1 billion per year. One of the main HAIs is caused by the endospore-forming bacterium Clostridium difficile. The most common cause of healthcare-acquired diarrhoea in the developed world, C. difficile was responsible for around 850 deaths in England and Wales in 2011. To help reduce the spread of the HAI-causing bacteria, terminal disinfection of isolation rooms and wards using hydrogen peroxide vapour is actively promoted. The key advantages of hydrogen peroxide vapour are its high oxidation potential which has been reported to inactivate bacteria, fungi and spores. An additional advantage of hydrogen peroxide vapour is that it is relatively environmentally friendly, breaking down into oxygen and water. Investigation into bacterial inactivation kinetics was undertaken at controlled, steady concentrations of hydrogen peroxide vapour in the range of 10 ppm to 90 ppm. An exposure chamber was designed whereby the bacterial spores could be exposed to constant concentrations of hydrogen peroxide for various exposure times. Bacterial spores (1-log10 to 8-log10 cfu) were filter deposited onto membranes to achieve an even layer for consistent exposure of the hydrogen peroxide vapour to the spores. Bacillus subtilis is often used for method development in bacterial studies; advantages are it has been shown to be highly resistant to hydrogen peroxide vapour and is not a human pathogen. Following the method development, different strains of C. difficile (ribotypes 014, 027, 103 and 220) were exposed to identify differences in resistance. Inactivation models (Chick-Watson, Series-Event, Weibull and Baranyi) were used to fit the data generated using the environmental chamber. Decimal reduction values (D-values) were calculated from the models for comparative studies regarding the inactivation achieved for the different bacteria and different hydrogen peroxide concentrations. The findings from this thesis revealed the Weibull model provides the best fit for most of the data. An initial shoulder period was identified for B. subtilis which was absent for C. difficile inactivation by hydrogen peroxide vapour; B. subtilis is therefore more resistant to hydrogen peroxide disinfection than C. difficile. Typical D-values for B. subtilis and C. difficile when exposed to hydrogen peroxide vapour at a concentration of 90 ppm were 140 and 1 min, respectively. C. difficile inactivation data were used to develop a model to estimate the log reduction that could be achieved during an inactivation cycle based on the concentration-time integral ( ). This model could be used to estimate the log reduction of commercially available hydrogen peroxide decontamination systems; these release a fixed amount of hydrogen peroxide into the room resulting in a peak concentration before decomposition to oxygen and water. Releasing the hydrogen peroxide into the room in this manner results in spatial and temporal variation; this could result in differences in bacterial inactivation in different areas within the room. Using the aforementioned regression model, the inactivation achieved at all locations within the room could be predicted, which could be used to optimise the current hydrogen peroxide decontamination cycles.

Loop-mediated isothermal DNA amplification (LAMP) are currently used as standalone diagnostic test for C. difficile infection (CDI). We assessed the diagnostic accuracy of LAMP for the diagnosis of CDI. We searched 5 databases to identify studies that compared LAMP with culture cytotoxicity neutralization assay or anaerobic toxigenic culture (TC) of C. difficile. We used the random-effects model to calculate pooled sensitivities, specificities, diagnostic odds ratios and their 95% confidence intervals (CIs). The search of the databases yielded 16 studies (6,979 samples) that met inclusion criteria. When TC was used as the gold standard (6,572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95%CI, 0.93-0.97; I2 = 67.4) and a mean specificity of 0.99 (95%CI, 0.96-1.00; I2 = 97.0). LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should however be interpreted only in the presence of clinical suspicion and symptoms of CDI. / Revisión por pares

<p><em>Clostridioides difficileinfection</em>  (CDI)  is  considered  as  an  urgent  threat  to  the  publicby  CDC, 2019.It  causes  life-threatening  diarrhea  and pseudomembranous colitis,mostly  in those  taking antibiotics or at the end of their antibiotic course.It is also notifiedas hospital-associated pathogensbecause one-third  of  the CDIhas occurredinthe health  care  center. Norepinephrine  (NE)  is  a stress-associated  neuroendocrine  hormone  released  upon  sympathetic  stimulation  to  mediate stress.Gut walls are highly innervated by the sympathetic nervous system. During stress, elevated level  of  NE  released  in  the  GI  tractcould  influence  bacterial  overgrowth  &  translocation.  It  isalready known  for  its  role  in  modulating  the  behavior  of  several  bacterial  pathogens  suchas Staphylococcus, Escherichia coli, Salmonella, and Vibrio cholera. This study aims to evaluate the effect of NE treatment on the growth and virulence of C. difficile.Here, we studied the effect of NE  on  six  different C.  difficilestrains  isolated  from humans. To  understandthe  influence  on growth, bacterial culture was treated (+/-)NE (5μM & 50 μM)during their log phase and recorded the density of the cell each time period for constructing the growth curve. In addition, after NE treatment, bacterial cells were taken for further analysis. For investigating the impact of NE on the virulence  genes  expression, a qPCR  reaction  was  performed  along  with -RT  / noRT  control reactions  for assessingthe  RNA  sample  free  from  genomic  DNA  contamination.  In the case of growth,higher growth was observed in VPI 10463at 6 hourtime pointonly,and in strain,NR 49277 significantly stimulated after 6 hoursand continued till 8 hours after treatmentwith50μM NE. In strain NR 49282, decreasedgrowth was observed at7-hourtime pointsafter 50 μM NEtreatment.But, there was no difference in cell density between control &  5μM NE treated bacterial culture in all strains.</p> <p>Toxingenes(tcdA&tcdB)and flagellin gene(fliC),were upregulated in NR 49290, NR 49277 & VPI 10463strains in both concentrations of NE and down-regulated in NR 49282.In strain NR 32888, toxin genes were downregulated while treated with 5μM NEbut upregulated after 50μM NEtreatment, though fliC was downregulated in both concentrations. In strain NR 32891,  tcdAwas downregulated,but tcdB& fliCwere upregulatedafter NE treatmentin both concentrations. Increased expression in pilin gene,pilA1in strain NR 49277, NR 49290, VPI 10463& NR 32891 in both concentrationswas observed.  In addition, pilA3in NR 49277, VPI 10463& NR 32891 and PilA5in  NR  49277  &  NR  49290  showed an upregulation pattern while  treated  with  both concentrations. Modulating this response, it is possible to reduce the pathogenicity of C. difficileduring medical care & antibiotic use.</p> <p><br></p>

C. difficile est une bactérie anaérobie du tube digestif reconnue comme l’agent responsable de 25% des diarrhées nosocomiales post antibiotiques et de la plupart des colites pseudomembraneuses. Le métronidazole et la vancomycine sont les traitements de référence pour les infections liées à ce pathogène. A ce jour, aucune souche de C. difficile résistante à la vancomycine n’a été rapportée. Paradoxalement, 85 % des souches de cette espèce hébergent dans leur génome un groupe de gènes cryptiques vanGCd homologue à l’opéron de résistance à la vancomycine vanG de E. faecalis. Dans ce travail, nous avons étudié la capacité des gènes vanGCd à conférer la résistance à la vancomycine. L’étude transcriptionnelle a révélé que le groupe de gènes vanGCd est transcrit en deux opérons (i) l’un de régulation exprimé de façon constitutive et (ii) l’autre de résistance, inductible par la vancomycine. L’analyse enzymatique de VanGCd, VanXYCd et VanTCd in vitro a montré que ces protéines possèdent respectivement des activités ligase, D,D-peptidase et racémase nécessaires au pontage D-Ala-D-Ser et à l’hydrolyse des dipeptides naturels D-Ala-D-Ala. L’analyse des précurseurs du peptidoglycane par spectrométrie de masse en présence de vancomycine a permis l’identification de l’UDP-MurNAc-pentapeptide [Ser] témoin de l’activité in vivo de ces protéines. L’opéron de résistance vanGcd cloné chez E. coli NR698 sensible à la vancomycine exerce un très faible effet sur la CMI de cet antibiotique. En revanche, le clonage chez E. faecalis n’a pas été obtenu en raison de mutations spontanées. Pour ces raisons, nous avons introduit l’opéron vanC de E. gallinarum chez C. difficile et observé une faible augmentation de la CMI de la vancomycine. Ce résultat indique que l’expression de la résistance à cet antibiotique chez C. difficile implique des facteurs intrinsèques liés à la bactérie. Nous avons observé que la ligase MurF possède une meilleure affinité vis-à-vis du dipeptide D-Ala-D-Ala que pour le dipeptide D-Ala-D-Ser ce qui pourrait impacter la synthèse de l’UDP-MurNAc-pentapeptide[Ser]. Par ailleurs, nous avons observé qu’en présence de vancomycine, les précurseurs du peptidoglycane sont amidés. Cette modification affecte également le peptidoglycane mature. Toutefois, le rôle physiologique de l’amidation chez C. difficile reste à élucider. En conclusion, (i) l’absence d’expression de la résistance à la vancomycine de C. difficile est multifactorielle, (ii) le groupe de gènes vanGCd a peu de potentiel pour contribuer à l’émergence de la résistance à la vancomycine. / Clostridium difficile is a major enteric pathogen responsible for 25% of post antibiotics diarrhea and most cases of pseudomembranous colitis. Metronidazole and vancomycin are the standard treatments for infected patients. vanGCd, a cryptic gene cluster highly homologous to the vanG gene cluster of Enterococcus faecalis is largely spread in Clostridium difficile. Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanGCd cluster to confer vancomycine resistance. We showed that expression of vanGCd is inducible by vancomycin and that VanGCd, VanXYCd and VanTCd are functional, exhibiting D-Ala:D-Ser ligase, D,D-dipeptidase and D-Ser racemase activities, respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans-complementation of C. difficile with the vanC resistance operon of Enterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C. difficile. Sub-lethal concentration of vancomycin led to production of UDP-MurNAc-pentapeptide [D-Ser], suggesting that the vanGCd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP-MurNAc-tetrapeptide, UDP-MurNAc-pentapeptide[D-Ala] and UDP-MurNAc-pentapeptide[D-Ser]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra-Tetra is amidated on the meso-diaminopimelic acid. We also demostrated that the ligase MurF has a better affinity for the D-Ala-D-Ala dipeptide than for the D-Ala-D-Ser, which could impact on the synthesis of UDP-MurNAc-pentapeptide [Ser].In conclusion, the lack of vancomycin resistance expression may be due to several factors that, in combinination with a gene cluster conferring a low level vancomycin resistancemay prevent the emergence of vancomycin resistance based on D-Ala- D-Ser modification of peptidoglycane precursors in C. difficile.

Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

Hospital-associated infections (HAIs) are infections patients contract as a result of being hospitalized. HAI rates decreased for almost all pathogens in the past few years, with the exception of Clostridium difficile infections (CDIs), which have been steadily climbing, placing hospital-acquired CDI at the top of the HAI list. The Center for Disease Control and Prevention reported in 2010 almost a half a million people were infected with CDIs yearly in the United States, and CDIs claimed the lives of approximately 29,000 people, representing a 4-fold increase from 1993. To address the problem in the local hospital, a quality improvement initiative called Bleach-It-Away was initiated. The initiative involved nurses wiping down the high touch areas in the patient's medical intensive care (MICU) rooms once every shift. The purpose of this quantitative research project was to evaluate the effectiveness of the Bleach-It-Away practice. The project question asked if the Bleach-It-Away practice was effective in reducing CDI rates. Deidentified CDI rates were provided by the clinical practice site covering a period of 12 months prior to implementation and 12 months after implementation of the practice. An independent t-test was used to determine whether there were significant improvements in CDI rates in the MICU. No significant improvement was seen in the postimplementation total CDI rates (p=.07) compared to the preimplementation rates. While the process did not demonstrate a significant improvement, positive social change is possible as hospitals recognize the many factors contributing to CDIs and the need for collaboration from various disciplines to control the problem.

No description available.

Clostridium difficile est responsable de vingt-cinq pour cent des diarrhées post-antibiotiques et de la majorité des cas de colite pseudomembraneuse. C'est un bacille anaérobie à Gram positif sporulant. La bactérie est recouverte par un réseau cristallin bidimensionnel appelé couche S. Elle est formée par deux sous-unités de haut et bas poids moléculaire issues du clivage du précurseur SlpA par la protéase Cwp84. Ces deux protéines sont codées par des gènes situés dans le locus cwp et sont sécrétées par le système de sécrétion de type SecA2. Elles sont impliquées dans l'adhésion/colonisation du côlon par C. difficile. L'adhésion est une étape commune avec la formation du biofilm par les bactéries. Le biofilm est une communauté bactérienne enchâssée et protégée par une matrice extracellulaire produite par les membres de la communauté. C'est le mode de vie principal des bactéries. Nous avons caractérisé la voie de sécrétion de type SecA2 de C. difficile. La protéine SecA2 (codée également par un gène du locus cwp) est essentielle pour la survie de C. difficile. Elle a une double localisation : le cytoplasme et la membrane intracellulaire. SecA2 de B. anthracis est capable de dimériser avec les protéines SecA1 et SecA2 de C. difficile. De plus, la complémentation du mutant secA2 de B. anthracis par le gène secA2 de C. difficile est fonctionnelle. Le rôle de la couche S, de la protéase Cwp84 et de la mobilité dans le biofilm de C. difficile ont été également étudiés. La souche 630∆erm forme un biofilm fin et fragile tandis que le mutant 630∆ermcwp84::erm forme un biofilm épais et robuste. Nous avons montré que l'activité protéolytique de Cwp84 était impliquée dans la formation du biofilm. De plus, une inhibition de la traduction de l'ARN slpA avec un ARN antisens spécifique permet une augmentation de la taille du biofilm formé par la souche 630∆erm. Similairement, l'expression de l’allèle secA2 muté dominant qui bloque au moins partiellement la voie de sécrétion de type SecA2 augmente la taille du biofilm. Nos résultats suggèrent que la couche S, la protéase Cwp84 et la protéine SecA2 sont impliquées dans la formation du biofilm de C. difficile. Par ailleurs nous avons testé un panel de souches dans leur capacité à former un biofilm. Les résultats montrent que les souches non mobiles ne seraient pas capables de former un biofilm épais. Enfin, nous avons étudié la capacité de C. difficile à se développer en aérobiose au sein d’un biofilm mixte avec Bacillus cereus. Nous avons mis en évidence un recrutement et une multiplication de C. difficile dans la pellicule formée à l'interface air/liquide par B. cereus. Un rapport optimal des spores des deux espèces est requis pour le développement de C. difficile dans ces conditions. La présence de spores de C. difficile dans la pellicule suggère que les biofilms de l'environnement pourraient être des réservoirs de spores de C. difficile, et à l'origine de contaminations humaine et animale. / Clostridium difficile is responsible for twenty-five percent of post-antibiotics diarrhea and for most cases of pseudomembranous colitis. It is an anaerobic, sporulating, Gram-positive bacillus. The bacterium is covered by a two-dimensional lattice called S-layer. It is formed by two subunits of high and low molecular weight after the cleavage of the SlpA precursor by the Cwp84 protease. These two proteins are encoded by genes located in the locus cwp and are secreted by the SecA2 secretion system. They are involved in colonic adhesion/colonization by C. difficile. Adhesion is a common step with biofilm formation by bacteria. The Biofilm is a microbial community embedded in and protected by an extracellular matrix produced by the community members. The biofilm is the main bacterial way of life.We have characterized the SecA2 secretory pathway of C. difficile. The SecA2 protein (as encoded by a gene locus cwp) is essential for the survival of C. difficile. It has a dual location: cytoplasm and intracellular membrane. SecA2 of B. anthracis is able to dimerize with SecA1 and SecA2 proteins of C. difficile. Moreover, the complementation of B. anthracis secA2 mutant with secA2 gene of C. difficile is functional.The role of the S-layer, of the Cwp84 protease and of the motility in the biofilm of C. difficile have also been studied. The 630∆erm strain forms a thin and weak biofilm while the 630∆ermcwp84::erm mutant forms a thick and robust biofilm. We have shown that proteolytic activity of Cwp84 was involved in biofilm formation. Furthermore, a decrease in the translation of slpA RNA with an antisense RNA specific permits an increase in the size of the biofilm of the strain 630∆erm strain. Similarly, the expression of the dominant mutated secA2 allele, which at least partially blocks the SecA2 secretory pathway, increases the biofilm size. Our results suggest that the S-layer, the Cwp84 protease and the SecA2 protein are involved in biofilm formation of C. difficile. On the other hand, we have tested a panel of strains in their capacity to form a biofilm. The results show that non-motile strains are unable to form a thick biofilm.Finally, we have studied the ability of C. difficile to grow aerobically in a mixed biofilm with B. cereus. We highlighted a recruitment and proliferation of C. difficile in the film formed at the air/liquid interface with B. cereus. An optimal ratio of spores of both species is required for the development of C. difficile in these conditions. The presence of C. difficile spores in the film suggests that the environment biofilms could be reservoirs of spores of C. difficile, and the source of human and animal contamination.