Dorota Wultańska

Clostridioides difficile, the causative agent of C. difficile infections (CDI), can be naturally infected by bacterial viruses known as bacteriophages. All characterized bacteriophages of this bacterium are temperate, meaning that upon infection their genetic material integrates and replicates with host’s genome. Such lysogenic strains can exhibit altered physiology and virulence, which in turn can be an important factor for epidemiology of CDI. In this study we characterized the phiCDKH02 bacteriophage infecting clinical isolates of C. difficile belonging to hypervirulent ribotypes 027 and 176. The bacteriophage was found to be identical to phi027. To get some insight into the role of this bacteriophage in physiology of its host and interaction with human colon cells, we made use of CRISPR-Cpf1 technology to cure the lysogenic C. difficile of the prophage. The prophage-free strain exhibited altered sporulation efficiency, lowered adhesion and decreased cytopathic effects to-wards human colon cells. These results emphasize importance of prophages in shaping virulence of C. difficile.

Clostridioides difficile is a causative agent of antibiotic-associated diarrhea as well as pseudomembranous colitis. So far, all known bacteriophages infecting these bacteria are temperate, which means that instead of prompt lysis of host cells, they can integrate into the host genome or replicate episomally. While C. difficile phages are capable of spontaneous induction and entering the lytic pathway, very little is known about the regulation of their maintenance in the state of lysogeny. In this study, we investigated the properties of a putative major repressor of the recently characterized C. difficile phiCDKH01 bacteriophage. A candidate protein belongs to the XRE family and controls the transcription of genes encoding putative phage antirepressors, known to be involved in the regulation of lytic development. Hence, the putative major phage repressor is likely to be responsible for maintenance of the lysogeny.

Understanding the potential antimicrobial properties of natural compounds and their impacts on Clostridioides difficile virulence factors may aid in developing alternative strategies for preventing and treating C. difficile infections (CDI). In this study, we investigated the bactericidal effects of ginger oil (GO), peppermint oil (PO), curcumin (CU), cinnamon aldehyde (CI), and trans-cinnamaldehyde (TCI) on the adhesion and biofilm disruption of C. difficile. We used three reference and five clinical C. difficile strains of different ribotypes. The bactericidal activity was assessed using the broth microdilution method. The adhesion was evaluated using human epithelial cell lines, and biofilm formation was visualized by confocal laser scanning microscopy. All tested strains exhibited susceptibility to CU, with minimum inhibitory concentration (MIC) values ranging from 128 µg/mL to 2048 µg/mL. Similarly, all strains were susceptible to CI and TCI, with MIC values ranging from 6.25% (v/v) to 25% (v/v). Most of the tested substances reduced the adhesion of C. difficile strains, while two tested strains showed significantly higher adhesion when co-incubated with the tested substances. Similar observations were made for biofilm formation, with observed density and morphology varied depending on the strain. In conclusion, the tested products demonstrated bactericidal activity and reduced the adhesion of C. difficile strains. They may be considered for further studies as potential antimicrobial agents targeting biofilm-related infections.

Clostridioides difficile is a predominant nosocomial pathogen within the healthcare setting able to produce biofilms. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics trigger mechanisms affecting bacterial virulence, including increased adhesion and biofilm formation. The aim of this study was to investigate how sub-MICs of metronidazole affect the biofilm formation of C. difficile strains. We tested 14 reference and clinical C. difficile strains, including hypervirulent strains of RT027. The MICs of metronidazole for the tested strains were determined using the broth microdilution method. Biofilm formation was evaluated using confocal laser scanning microscopy. The C. difficile strains belonging to RT027 produced the highest amounts of biofilm. The results of confocal laser scanning microscopy showed that all the tested C. difficile strains developed larger biofilms with diversified architectures upon exposure to sub-MICs of metronidazole. In our study, we reveal that sub-MIC concentrations of metronidazole affect the biofilm formation of clinical and reference strains of C. difficile. Importantly, metronidazole induces biofilm formation via hypervirulent RT027 strains.

There is an ongoing search for alternative treatments for Clostridioides difficile infections. The aim of the study was to investigate the antibacterial and antibiotic activity of bee products against C. difficile strains with different polymerase chain reaction ribotypes (RTs). The minimum inhibitory concentration (MICs) of Manuka honey 550+, goldenrod honey, pine honey, and bee bread were determined by the broth dilution method. C. difficile adhesion to HT-29, HT-29 MTX, and CCD 841 CoN cell lines was assessed. Biofilm was cultured in titration plates and visualized by confocal microscopy. The MICs of Manuka honey for C. difficile 630 and ATCC 9689 strains and control strain, M 120, were 6.25%, 6.25%, and 1.56% (v/v), respectively; of goldenrod honey, 50%, 50%, and 12.5%, respectively; of pine honey, 25%, 25%, and 25%, respectively; and of bee bread, 100 mg/L, 50 mg/L, and 100 mg/L, respectively. Manuka honey (1%) increased adhesion of C. difficile RT176 strains, and one strain of RT023, to the CCD 841 cell line. Pine honey (1%) increased RT027 adhesion to the HT-29 cell line. Manuka honey, pine honey, and bee bread at subinhibitory concentrations increased the adhesion of C. difficile. Our research proved that bee products are active against the tested strains of C. difficile.

Aim: The objective of this study was to determine the effect of standard and candidate prebiotics on the adhesion and biofilm formation of Bacteroides sp. in monoculture and co-culture with Clostri...

Diarrhea is a common problem in nursing homes. A survey among nursing facilities in Poland was used to characterize diarrhea outbreaks, the burden caused for residents and caregivers and the employed measures. Survey results confirmed that diarrhea is a common problem in nursing homes and in most cases affects groups of residents. The related burden is high or very high for 27% of residents and 40% of caregivers. In 80% of nursing facilities pro or synbiotics are part of the measures used to manage diarrhea. Administration of these kinds of products has been suggested for the management of diarrhea, especially in cases caused by Clostridioides (C.) difficile. C. difficile is one of many potential causes for diarrhea, but is of particular concern for nursing homes because it is responsible for a large proportion of diarrhea outbreaks and is often caused by multi-drug resistant strains. In vitro inhibition of a quinolone-resistant and a multi-drug resistant C. difficile strain was used to evaluate the growth inhibitory effects of commonly used products containing probiotic microorganisms. Growth of both strains was best inhibited by multi-strain synbiotic preparations. These findings suggest that multi-strain synbiotics can be considered as an interventional option for diarrhea caused by C. difficile.

We aimed to describe the clinical presentation, treatment, outcome and report on factors associated with mortality over a 90-day period in Clostridioides difficile infection (CDI). Descriptive, univariate, and multivariate regression analyses were performed on data collected in a retrospective case-control study conducted in nine hospitals from seven European countries. A total of 624 patients were included, of which 415 were deceased (cases) and 209 were still alive 90 days after a CDI diagnosis (controls). The most common antibiotics used previously in both groups were β-lactams; previous exposure to fluoroquinolones was significantly (p = 0.0004) greater in deceased patients. Multivariate logistic regression showed that the factors independently related with death during CDI were older age, inadequate CDI therapy, cachexia, malignancy, Charlson Index, long-term care, elevated white blood cell count (WBC), C-reactive protein (CRP), bacteraemia, complications, and cognitive impairment. In addition, older age, higher levels of WBC, neutrophil, CRP or creatinine, the presence of malignancy, cognitive impairment, and complications were strongly correlated with shortening the time from CDI diagnosis to death. CDI prevention should be primarily focused on hospitalised elderly people receiving antibiotics. WBC, neutrophil count, CRP, creatinine, albumin and lactate levels should be tested in every hospitalised patient treated for CDI to assess the risk of a fatal outcome.