Associations of motility and auto-aggregation with biofilm-formation capacity levels in Clostridioidesdifficile.

Clostridioides difficile (C. difficile) is responsible for one of the most common nosocomial infections worldwide. This work assessed associations between biofilm-formation capacity levels of C. difficile and cell viability, motility, flagella, motility and auto-aggregation in 118 clinical isolates. Biofilm production was assessed by the crystal violet method. Cell viability was determined by BacTiter-Glo™ Microbial Cell Viability Assay and live-imaging microscopy. Expression levels of LuxS, Cwp84, Spo0A, PilA, and FliC were measured by real-time PCR. Motility was visually assessed in agar tubes. Auto-aggregation levels were determined by OD600 measurements. Out of 118 isolates, 66 (56 %) were biofilm producers, with most being strong or moderate producers. Cell viability, motility and auto-aggregation positively correlated with biofilm-production capacity (p = 0.0001, p = 0.036 and p < 0.0001, respectively). Positive associations were found between pilA, fliC and luxS expression levels and biofilm-production capacity (p = 0.04, p = 0.01, p = 0.036, respectively). This is the first report of associations between biofilm-formation capacity and cell viability, pilA, fliC, and luxS gene expression, auto-aggregation and motility. These correlations should be further explored to expand knowledge on the regulation of C. difficile biofilm formation, and pathogenesis, which will have notable implications on treatment options.

The effect of different C. difficile MLST strains on viability and activity of macrophages.

Objectives: Clostridioides difficile is the most common infectious agent of nosocomial diarrhea. C. difficile infection (CDI) pathogenesis and disease severity depend on its toxins (toxins A, B and binary) and on the host's immune response, especially the innate immune system. The current study examined the efficacy of macrophage activity, macrophages viability and cytokine secretion levelsin response to different sequence type (ST) strains of C. difficile. Methods: RAW 264.7 macrophages were exposed to six different strains of C. difficile as well as to both toxins A and B and macrophage viability was measured. The levels of four secreted cytokines were determined by RT-PCR and ELISA. Morphological changes to the macrophages were investigated by fluorescent microscopy. Results: Strains ST37 and ST42 affected macrophages' vitality the most. Toxins A and B led to a significant reduction in macrophages' vitality at most time points. In addition, starting at 30-min post-exposure to 5 ng/µl of both toxins led to significant differences in macrophage viability versus at lower concentrations. Furthermore, cytokine secretion levels, including IL-12, IL-6 and TNF-α, increased dramatically when macrophages were exposed to strains ST42 or ST104. Finally, gene expression surveys point to increases in IL-12 gene expression in response to both ST42 and ST104. Conclusions: C. difficile strains with higher toxins levels induced an increased activation of the innate immune system and may activate macrophages more profoundly resulting in secretion of higher levels of pro-inflammatory cytokines. However, higher toxin levels may also damage macrophages' normal skeletal structure, reducing macrophage viability.

Different bile acids have versatile effects on sporulation, toxin levels and biofilm formation of different Clostridioides difficile strains.

Clostridioides difficile infection develops following ingestion of virulent stains by a susceptible host. Once germinated, toxins TcdA and TcdB, and in some of the strains binary toxin, are secreted, eliciting disease. Bile acids play a significant role in the process of spore germination and outgrowth, with cholate and its derivative enhancing colony formation, while chenodeoxycholate inhibit germination and outgrowth. This work investigated bile acids' impact on spore germination, toxin levels and biofilm formation in various strain types (STs). Thirty C. difficile isolates (A+ B+ CDT-\+) of different STs were exposed to increasing concentrations of the bile acids, cholic acid (CA), taurocholic acid (TCA) and chenodeoxycholic acid (CDCA). Following treatments, spore germination was determined. Toxin concentrations were semi-quantified using the C. Diff Tox A/B II™ kit. Biofilm formation was detected by the microplate assay with crystal violet. SYTO® 9 and propidium iodide staining were used for live and dead cell detection, respectively, inside the biofilm. Toxins levels were increased by 1.5-28-fold in response to CA and by 1.5-20-fold in response to TCA, and decreased by 1-37-fold due to CDCA exposure. CA had a concentration-dependent effect on biofilm formation, with the low concentration (0.1%) inducing- and the higher concentrations inhibiting biofilm formation, while CDCA significantly reduced biofilm production at all concentrations. There were no differences in the bile acids effects on different STs. Further investigation might identify a specific bile acids' combination with inhibitory effects on C. difficile toxin and biofilm production, which could modulate toxin formation to reduce the likelihood of developing CDI.

The Cytopathic Effect of Different Toxin Concentrations From Different Clostridioides difficile Sequence Types Strains in Vero Cells.

Clostridioides difficile is one of the leading causes of healthcare-associated diarrhea, with severity ranging from mild, self-limiting disease, to life-threatening toxic megacolon. C. difficile infection (CDI) pathogenesis is mediated by the TcdA and TcdB toxins. This work aimed to draw correlations between toxin levels, bacterial strains, and disease severity in 63 CDI patients. C. difficile typing was performed by multi-locus sequence types (MLST). Toxin concentrations were measured using the TOX A/B test. In addition, cell cytotoxicity assay was performed following Vero cell exposure to stool extracts (24 h). The most prevalent sequence types (ST) were ST2, ST4, ST6, ST13, ST37, ST42, and ST104, with highest toxin levels produced by ST42 and ST104 (302.5 and 297.1 ng/ml, respectively). These strains had a stronger cytopathic effect (CPE) on Vero cells as compared to strains with lower toxin concentrations (p < 0.001), as manifested by lower cell counts and higher percentages of cell rounding and adhesion loss. Although no association was found between ST, toxin concentrations, and disease severity, a diverse in vitro effect of different STs on the viability and activity of Vero cells was observed. These findings suggest that disease severity is affected by both host immune responses and by bacterial characteristics.

IL-16 and BCA-1 Serum Levels Are Associated with Disease Severity of C. difficile Infection.

Clostridioides difficile infection (CDI) is associated with a high risk for complications and death, which requires identifying severe patients and treating them accordingly. We examined the serum level of six cytokines and chemokines (IL-16, IL-21, IL-23, IL-33, BCA-1, TRAIL) and investigated the association between them and patients' disease severity. Concentrations of six cytokines and chemokines were measured using the MILLIPLEX®MAP kit (Billerica, MA, USA) in serum samples attained from CDI patients within 24-48 h after laboratory confirmation of C. difficile presence. Demographic and clinical data were collected from medical records. The disease severity score was determined according to guidelines of the "Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America" (SHEA-IDSA). Out of 54 patients, 20 (37%) had mild to moderate disease and 34 (63%) had severe disease. IL-16 (p = 0.005) and BCA-1 (p = 0.012) were associated with a more severe disease. In conclusion, IL-16 and BCA-1, along with other cytokines and chemokines, may serve as biomarkers for the early prediction of CDI severity in the future. An improved and more accessible assessment of CDI severity will contribute to the adjustment of the medical treatment, which will lead to a better patient outcome.

The microbial diversity following antibiotic treatment of Clostridioides difficile infection.

BACKGROUND: Clostridioides difficile (C. difficile) is a major nosocomial pathogen that infects the human gut and can cause diarrheal disease. A dominant risk factor is antibiotic treatment that disrupts the normal gut microbiota. The aim of the study was to examine the correlation between antibiotic treatment received prior to C. difficile infection (CDI) onset and patient gut microbiota. METHODS: Stool samples were collected from patients with CDI, presenting at the Baruch Padeh Medical Center Poriya, Israel. Demographic and clinical information, including previous antibiotic treatments, was collected from patient charts, and CDI severity score was calculated. Bacteria were isolated from stool samples, and gut microbiome was analyzed by sequencing the 16S rRNA gene using the Illumina MiSeq platform and QIIME2. RESULTS: In total, 84 patients with CDI were enrolled in the study; all had received antibiotics prior to disease onset. Due to comorbidities, 46 patients (55%) had received more than one class of antibiotics. The most common class of antibiotics used was cephalosporins (n = 44 cases). The intestinal microbiota of the patients was not uniform and was mainly dominated by Proteobacteria. Differences in intestinal microbiome were influenced by the different combinations of antibiotics that the patients had received (p = 0.022) CONCLUSIONS: The number of different antibiotics administered has a major impact on the CDI patients gut microbiome, mainly on bacterial richness.

Reduced Susceptibility to Chlorhexidine among Staphylococcus aureus Isolates in Israel: Phenotypic and Genotypic Tolerance.

Antiseptic use for body decolonization is the main activity applied to prevent healthcare-associated infections, including those caused by S. aureus. Consequentially, tolerance to several antiseptics such as chlorhexidine gluconate (CHG) has developed. This study aimed to estimate the prevalence of CHG tolerance among S. aureus strains in Israel and to evaluate factors that may affect this tolerance. Furthermore, it tested the associations between phenotypic and genotypic CHG tolerance. S. aureus strains (n = 190) were isolated from clinical samples of patients admitted to various medical institutions in Israel. Phenotypic susceptibility to CHG was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Genotypic tolerance was detected using real-time PCR for detection of qac A/B genes. MIC for the antibiotic mupirocin was determined using the Etest method. Presence of the Panton-Valentine Leucocidin (pvl) toxin, mecA and mecC genes was detected using an eazyplex® MRSAplus kit (AmplexDiagnostics GmbH, Gars, Germany). CHG tolerance was observed in 13.15% of the isolates. An association between phenotypic and genotypic tolerance to CHG was observed. Phenotypic tolerance to CHG was associated with methicillin resistance but not with mupirocin resistance. Additionally, most of the CHG-tolerant strains were isolated from blood cultures. In conclusion, this work shed light on the prevalence of reduced susceptibility to CHG among S. aureus strains in Israel and on the characteristics of tolerant strains. CHG-tolerant strains were more common than methicillin-resistant ones in samples from invasive infections. Further research should be performed to evaluate risk factors for the development of CHG tolerance.

The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay.

This research compares the performance and diagnostic accuracy of three molecular tests for the detection of Clostridium difficile in stool samples, with the Xpert® C. difficile assay. Fifty-nine positive and twenty-five stool negative samples were analyzed by the BDmax™ Cdiff, the Simplexa C. difficile Direct, and the GenomEra™ C. difficile, and compared with the Xpert® C. difficile routinely used in our laboratory. The highest sensitivity was 94.9% for the BDmax™ Cdiff, followed by GenomEra™ C. difficile with 93.2%, and Simplexa™ C. difficile Direct with 89.8%. The specificities of all assays were 100%. GenomEra™ C. difficile had the highest retesting rate (12%). Simplexa™ C. difficile Direct benefits from both short hands-on time and total-turnaround time and 0% retesting. The differences in performance and accuracy between these three molecular assays are insignificant and all may be used as part of the routine algorithm for detection of C. difficile.

Characterization of the Immune Response during Infection Caused by Clostridioides difficile.

The high risk of complications and death following Clostridioides difficile infection (CDI) requires identifying patients with severe disease and treating them accordingly. We characterized the immune response of CDI patients in relation to infection severity. Concentrations of 28 cytokines and chemokines were measured in serum samples, obtained from 54 CDI patients within a median timeframe of 24-48 h after laboratory confirmation of C. difficile infection. Demographic and clinical data were retrospectively collected from medical records. Disease severity score was determined by "Score indices for Clostridioides difficile infection severity". Of 54 patients (mean age, 76.6 years, 61.1% female), 38 (70.4%) had mild disease and 16 (29.6%) had moderate disease. Seven cytokines were associated with a more severe CDI: granulocyte-macrophage colony-stimulating factor (p = 0.0106), interleukin (IL)-1ß (p = 0.004), IL-8 (p = 0.0098), IL-12p70 (p = 0.0118), interferon-α (p = 0.0282), IL-15 (p = 0.0015), and IL-2 (p = 0.0031). Additionally, there was an increased T-helper 1 response in more severe cases of CDI. Cytokines may serve as biomarkers for early prediction of CDI severity. Better and earlier assessment of illness severity will contribute to the adjustment of medical treatment, including monitoring and follow-up.

Molecular Characterization and Moxifloxacin Susceptibility of Clostridium difficile.

In recent years, the incidence and severity of Clostridium difficile infections has increased.Additionally, resistance of C. difficile to frequently used antibiotics is rising. To improve ourunderstanding of C. difficile, there is a need for molecular characterization of different strains andantibiotic resistance testing. We investigated the efficacy of GenoType CDiff kit (Hain Lifesciences)in identification of C. difficile and its various strains in northern Israel. The kit involves a molecularassay that detects C. difficile from stool samples or colonies and identifies the different strains andmutations in the gyrA gene that cause moxifloxacin resistance. Forty-nine C. difficile positive sampleswere examined by the kit following DNA extraction from both colonies and stool. The identificationrate (95.9%) of C. difficile was much higher when DNA was extracted from colonies, compared toextraction from stool (46.9%). Low frequencies of ribotype027 strain (2%) and of ribotype078 strain(4%) were found. There was a high concordance between genotype (mutation in gyrA) andphenotype (Etest) for moxifloxacin resistance (Kappa=0.72). A high percentage of moxifloxacinresistantstrains was found. Our findings indicate that the GenoType CDiff kit is very effective incharacterization of C. difficile strains and less effective for identification of C. difficile directly fromstool samples.

Elevated serum immunoglobulin A levels in patients with Clostridium difficile infection are associated with mortality.

The current research investigated immunoglobulin A levels in serum of patients with Clostridium difficile infection. Our main aim was to test whether immunoglobulin A (IgA) levels at the early stage of infection can predict disease severity of C. difficile infection. Fifty-four patients were enrolled in the study. Stool samples were collected and analyzed by PCR for detection of C. difficile. IgA levels were measured in serum samples that were collected from each patient immediately after receiving a positive PCR result. Additionally, data concerning epidemiologic and clinical characteristics of the patients was retrospectively collected from the medical records. Serum IgA levels were not associated with the severity of C. difficile infection. However, a positive correlation between IgA levels and mortality during hospitalization was found. Additionally, IgA levels correlated with C-reactive protein and white blood cell levels. Although we did not find a correlation between IgA levels and C. difficile infection severity, we could associate IgA levels with the severity of general immune response and to complications of the infection, i.e. mortality. We think that this could represent a compensatory mechanism to the damage caused by C. difficile. Considering serum IgA anti-inflammatory functions, it is possible the increase in IgA levels aimed to suppress the more intense inflammatory response in patients who died during hospitalization.

In Vitro Activity of Tedizolid, Dalbavancin, and Ceftobiprole Against Clostridium difficile.

Background:Clostridium difficile (C. difficile) is a major nosocomial pathogen that colonizes in the human gut. Recently, the U.S. FDA approved three new antimicrobial agents against gram-positive bacteria: Tedizolid, Dalbavancin, and Ceftobiprole. The efficacy of these antibiotics for treatment of C. difficile infection has not been thoroughly examined. The current study aimed to examine the in vitro activity of these antibiotics against C. difficile. In addition, to compare between Dalbavancin and Ceftobiprole to antibiotics from the same class: Vancomycin and Ceftriaxone, respectively. Methods: Eighty-four C. difficile isolates were tested for susceptibility to Tedizolid, Dalbavancin, Ceftobiprole, Vancomycin, and Ceftriaxone by Etest technique in order to determine the minimum inhibitory concentration (MIC). Results: Upon comparison of the novel antibiotic agents, Dalbavancin demonstrated the lowest MIC values and ceftobiprole the highest at MIC50 (0.016, 0.38, and 1.5 µg/mL, for Dalbavancin, Tedizolid, and Ceftobiprole, respectively) and MIC90 (0.03, 0.78, and 3.17 µg/mL, respectively). Dalbavancin demonstrated significantly lower MIC50 and MIC90 values compared to Vancomycin (0.016 vs. 0.38 and 0.03 vs. 3.5, respectively) (p < 0.001) and ceftobiprole had significantly lower MIC values compare to ceftriaxone (1.5 vs. 32 and 3.17 vs. 28.8, respectively) (p < 0.001). Conclusion: Dalbavancin and Tedizolid may play a role as potential therapeutic agents for treatment of C. difficile infection. Examination of antibiotic effect on the intestinal microbiome and clinical trials are needed for more accurate results.

Role of Single Procalcitonin Test on Admission as a Biomarker for Predicting the Severity of Clostridium difficile Infection.

Objective: To evaluate whether serum Procalcitonin (PCT) at the early stage of infection can serve as a potential biomarker for determining Clostridium difficile infection (CDI) severity. Methods: Fifty-four patients diagnosed with CDI were enrolled in the study. Serum samples were obtained within a median time of 24-48 h of the lab result for presence of C. difficile. PCT levels were measured by chemiluminescence immunoassay. Demographic, clinical, and prognostic data concerning the patients were retrospectively collected from medical records. The illness severity score was determined according to "Score indices for C. difficile infection severity." Results: We found that serum PCT levels were significantly higher in patients with moderate disease, compared to patients with mild disease (p = 0.0032). Additionally, PCT was correlated with mortality (p = 0.0002), white blood cell count (p = 0.019), and community-acquired disease (p = 0.0345). Conclusion: Early measurement of PCT may serve as a biomarker for early prediction of CDI severity, which is of great importance due to the high risk of complications and death.