The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro.

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila-isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 µg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 µg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1ß, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier-related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells.

Alterations in the gut microbiota and their metabolites in human intestinal epithelial cells of patients with colorectal cancer.

BACKGROUND: The gut microbiota has become one of the main risk factors for the formation and development of colorectal cancer (CRC). CRC intensification may be due to the microbial pathogens' colonization and their released metabolites. Here, we analyzed Bacteroidetes and Clostridia bacteria in CRC patients and studied bacterial metabolome in cancerous tissues compared to their adjacent normal tissues. METHODS AND RESULTS: The population of selected bacteria in biopsy specimens of 30 patients with CRC was studied by RT-qPCR. The mutagenicity and cytotoxicity effects of microbiota metabolites were evaluated by Ames test and MTT Assay, respectively. Moreover, gene expression in carcinogenic pathways was studied by RT-qPCR, and genes with different expressions in tumor and non-tumor tissues were diagnosed. Based on microbiota analysis, the relative abundance of Clostridia and C. difficile was significantly higher in CRC tissue, whereas C. perfringens showed higher relative abundance in normal tissue. AIMES test confirmed the proliferation and mutagenicity effects of the bacterial metabolites in CRC patients. Significant upregulation of C-Myc, GRB2, IL-8, EGFR, PI3K, and AKT and downregulation of ATM were observed in CRC samples compared to the control. CONCLUSIONS: The influence of bacterial metabolites on inflammation and altered expression of genes in the cell signaling pathways was observed. The findings confirm the role gut microbiota composition and bacterial metabolites as key players in CRC onset and development.

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells.

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

Characterization of the mucosal microbiota in patients with nodular lymphoid hyperplasia with concurrent irritable bowel syndrome compared to healthy controls.

BACKGROUND: Nodular lymphoid hyperplasia (NLH) is known as a lymphoproliferative lesion in which multiple small nodules appear on the intestinal wall. It has been documented that patients who struggle with irritable bowel syndrome (IBS) are at greater risk of developing NLH. Here, we aimed to investigate the previously reported pathogens and the abundance of a selection of mucosal microbiota in IBS + NLH patients compared to IBS, and healthy controls. METHODS AND RESULTS: Terminal ileum biopsies were collected from 37 IBS + NLH, 37 IBS, and 29 healthy controls. Bacterial culture and PCR was performed to detect the presence of pathogens in biopsies. A qPCR assay was applied to assess the abundance of a selection of bacterial taxa. Totally, five bacterial isolates including two enteropathogenic and one enteroaggregative Escherichia coli (EPEC, EAEC), one enterotoxigenic Staphylococcus aureus (SEA), and one Yersinia enterocolitica strains were detected among the IBS + NLH cases. The relative abundance of Bacteroidetes and Streptococcus spp. in IBS + NLH patients was significantly less than IBS and healthy controls. Firmicutes, Pseudomonas spp., Haemophilus spp., and Campylobacter spp. were notably more abundant in IBS + NLH than in IBS patients. The abundance of Verrucomicrobia was higher in NLH + IBS than in healthy controls. Actinobacteria was also significantly more abundant among NLH + IBS patients than the controls. CONCLUSION: Our results demonstrated that mucosal microbiota composition in NLH + IBS patients slightly differs from that of IBS patients and healthy controls. Further research using large-scale cohorts are needed to enhance current understanding of the contribution of the mucosal microbiota to NLH pathogenesis with concurrent IBS.

Characterization of vaginal Lactobacillus species as a predictor of fertility among Iranian women with unexplained recurrent miscarriage and fertile women without miscarriage history using machine learning modeling.

BACKGROUND: Lactobacillus spp. are the predominant bacteria of the vaginal tract, the alteration of which has been previously linked to miscarriage. Here, we investigated differences between selected vaginal Lactobacillus species of women with a history of recurrent miscarriages and fertile women without a history of miscarriage in Iran. METHODS AND RESULTS: Vaginal swabs were taken from 29 fertile and 24 infertile women and quantitative real-time PCR (qPCR) assay was used to determine a selection of vaginal Lactobacillus species in both groups. The logistic regression (LR) model, Naive Bayes (NB) model, support vector machine model (SVM), and neural network model (NN) were developed to predict disease outcome by selected variables. LR analysis was used to construct a nomogram indicating predictions of the risk of miscarriage. The most abundant species among the patients were L. rhamnosus, L. ruminis, and L. acidophilus, while L. gasseri, L. vaginalis, L. fermentum, and L. iners were more abundant in healthy subjects. The distribution of L. ruminis, L. iners, and L. rhamnosus was higher in patients, while L. acidophilus, L. gasseri, and L. fermentum were highly distributed among healthy subjects. Higher AUC in predicting the disease outcome was observed for L. gasseri, L. rhamnosus, L. fermentum, and L. plantarum. CONCLUSION: Our findings provide experimental evidence of vaginal Lactobacillus imbalance in infertile women and a suitable predictor for miscarriage based on the AUC algorithms. Further studies with larger sample size and using high-throughput technologies are needed to boost our understanding of the role of lactobacilli in miscarriage.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells.

BACKGROUND: Surface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1ß and TNF-α cytokines was measured by ELISA. RESULTS: C. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-ß as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05). CONCLUSION: The results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Surface layer protein A from hypervirulent Clostridioides difficile ribotype 001 can induce autophagy process in human intestinal epithelial cells.

Clostridioides difficile is the leading cause of nosocomial diarrhea with high morbidity and mortality worldwide. C. difficile strains produce a crystalline surface layer protein A (SlpA), which is an absolute necessity for its pathogenesis. However, its pathogenic mechanisms and its pro-inflammatory behavior are not yet fully elucidated. Herein, we report for the first time that SlpA extracted from C. difficile can induce autophagy process in Caco-2 cells. SlpA protein was purified from two C. difficile strains (RT001 and ATCC 700075). The cell viability of Caco-2 cells after exposure with different concentrations (15, 20, 25 µg/mL) of SlpA at various time points (3, 6, 12, 24 h) was measured by MTT assay. Acridine orange staining was used to visualize the hypothetical acidic vesicular organelles. The gene expression of autophagy mediators including LC3B, Atg5, Atg16L, and Beclin-1 was determined by quantitative real-time PCR assay. Western blotting assay was used to detect the expression of LC3B protein. MTT assay showed that different concentrations of SlpA did not induce significant changes in the viability of Caco-2 cells. SlpA at concentration of 20 µg/mL enhanced the formation of acidic vesicular organelles in Caco-2 cells after 12 h of exposure. Moreover, SlpA treatment significantly increased the expression of autophagy-associated genes, and increased the expression of LC3B protein in Caco-2 cells. In conclusion, our study demonstrated that SlpA is capable to induce autophagy in intestinal epithelial cells. These findings reveal a novel mechanism for the pathogenesis of C. difficile mediated by its SLPs.

Curcumin and capsaicin regulate apoptosis and alleviate intestinal inflammation induced by Clostridioides difficile in vitro.

BACKGROUND: The dramatic upsurge of Clostridioides difficile infection (CDI) by hypervirulent isolates along with the paucity of effective conventional treatment call for the development of new alternative medicines against CDI. The inhibitory effects of curcumin (CCM) and capsaicin (CAP) were investigated on the activity of toxigenic cell-free supernatants (Tox-S) of C. difficile RT 001, RT 126 and RT 084, and culture-filtrate of C. difficile ATCC 700057. METHODS: Cell viability of HT-29 cells exposed to varying concentrations of CCM, CAP, C. difficile Tox-S and culture-filtrate was assessed by MTT assay. Anti-inflammatory and anti-apoptotic effects of CCM and CAP were examined by treatment of HT-29 cells with C. difficile Tox-S and culture-filtrate. Expression of BCL-2, SMAD3, NF-κB, TGF-ß and TNF-α genes in stimulated HT-29 cells was measured using RT-qPCR. RESULTS: C. difficile Tox-S significantly (P < 0.05) reduced the cell viability of HT-29 cells in comparison with untreated cells. Both CAP and CCM significantly (P < 0.05) downregulated the gene expression level of BCL-2, SMAD3, NF-κB and TNF-α in Tox-S treated HT-29 cells. Moreover, the gene expression of TGF-ß decreased in Tox-S stimulated HT-29 cells by both CAP and CCM, although these reductions were not significantly different (P > 0.05). CONCLUSION: The results of the present study highlighted that CCM and CAP can modulate the inflammatory response and apoptotic effects induced by Tox-S from different clinical C. difficile strains in vitro. Further studies are required to accurately explore the anti-toxin activity of natural components, and their probable adverse risks in clinical practice.

Involvement of Pseudomonas aeruginosa in the occurrence of community and hospital acquired diarrhea, and its virulence diversity among the stool and the environmental samples.

Transmission of Pseudomonas aeruginosa along the food chain could cause gastrointestinal infections. To show this involvement, the prevalence, putative virulence genotype, and antibiotic resistance phenotype of P. aeruginosa isolates from stool of 1482 patients with community and hospital acquired diarrhea were compared with 87 isolates from the environmental samples. The results showed infection with P. aeruginosa in 3.4% of the cases, while 57.4% of vegetable samples were contaminated. Significantly higher frequency of lasB (98%), aprA (98%), exoY (98%), and exoS (90%), but lower rate of exoT (39.2%), was detected among the stool isolates. Multi-drug resistance (MDR) phenotype was detected in 25.5% and 4% of the stool and vegetable isolates, respectively. A higher rate of studied virulence genes was detected among the MDR strains vs non-MDR strains. These results indicate P. aeruginosa as a causative agent of diarrhea either among the hospitalized patients and those with community-acquired diarrhea.

Serum procalcitonin levels associate with Clostridioides difficile infection in patients with inflammatory bowel disease.

BACKGROUND: Clostridioides difficile infection (CDI) is a major cause of morbidity among patients with inflammatory bowel disease (IBD). Diagnostic biomarkers for early detection of CDI are needed in clinical practice. The relationship between serum procalcitonin and CDI in IBD patients has not been investigated so far. Therefore, we aimed to evaluate the usefulness of measuring serum procalcitonin level to detect CDI in patients with the flare of IBD. METHODS: One hundred twenty patients with IBD were enrolled in this study. Bacterial identification was performed using standard microbiological and molecular methods. The serum procalcitonin levels were measured in all patients. Receiver operating characteristic (ROC) curve analysis was applied to assess the value of procalcitonin for the prediction of CDI among IBD patients. RESULTS: The median serum procalcitonin level was significantly increased in IBD patients with CDI compared to non-CDI IBD patients (0.69 ng/mL vs 0.32 ng/mL). In univariate analysis, log10 procalcitonin was associated with CDI (OR 2.81, 95% CI 1.54-4.09, P-value < 0.001). Procalcitonin 1.1 ng/mL was 85% sensitive and 88% specific for the prediction of CDI. In the multivariable model including the covariates log10 procalcitonin, age, hospitalization, type of IBD, duration of the disease, and antibiotic usage, procalcitonin showed a robust association with CDI (OR 4.59, 95% CI 2.49-6.70, P-value < 0.001). An elevated procalcitonin level was associated with the presence of CDI among IBD patients. CONCLUSIONS: Our results indicate that procalcitonin level can be a good candidate biomarker for assessing the CDI in IBD patients. Further studies are required to decipher whether procalcitonin can predict CDI therapy or its recurrence.

Characterization of the gut microbiota in patients with primary sclerosing cholangitis compared to inflammatory bowel disease and healthy controls.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease. Its etiology remains largely unknown, although frequent concomitant inflammatory bowel disease (IBD) hints towards common factors underlying intestinal and bile duct inflammation. Herein, we aimed to explore the relative abundance of fecal microbiota in PSC-IBD patients compared to IBD-only subjects and controls. METHODS AND RESULTS: We included 14 PSC-IBD patients, 12 IBD-only patients, and 8 healthy controls (HCs). A quantitative real-time PCR (qPCR) assay was used to determine a selection of bacterial phyla, families, and genera. Relative abundance of taxa showed that Bacteroidetes was the most abundant phylum among the patients with PSC-IBD (29.46%) and also HCs (39.34%), whereas the bacterial species belonging to the phylum Firmicutes were the most frequent group in IBD-only subjects (37.61%). The relative abundance of the Enterobacteriaceae family in fecal samples of PSC-IBD patients was similar to those with IBD-only, which was significantly higher than HCs (p value = 0.031), and thus, could be used as a PSC-IBD or IBD-only associated microbial signature. CONCLUSIONS: Our findings showed that intestinal microbiota composition in PSC-IBD patients was completely different from that of IBD-only patients. Further studies using large-scale cohorts should be performed to better describe the contribution of the gut microbiota to PSC pathogenesis with underlying IBD.

Genetic diversity and phylogenetic analysis of the surface layer protein A gene (slpA) among Clostridioides difficile clinical isolates from Tehran, Iran.

Clostridioides difficile is the most common causative agent of healthcare-associated diarrhea. C. difficile strains produce a crystalline surface layer protein (SlpA), encoded by the slpA gene. Previous studies have shown that SlpA varies among C. difficile strains. In this study, we used the SlpA sequence-based typing system (SlpAST) for the molecular genotyping of C. difficile clinical isolates identified in Iran; the PCR ribotypes (RTs) and toxin profiles of the isolates were also characterized. Forty-eight C. difficile isolates were obtained from diarrheal patients, and characterized by capillary electrophoresis (CE) PCR ribotyping and the detection of toxin genes. In addition, the genetic diversity of the slpA gene was investigated by Sanger sequencing. The most common RTs were RT126 (20.8%), followed by RT001 (12.5%) and RT084 (10.4%). The intact PaLoc arrangement representing cdu2+/tcdR+/tcdB+/tcdE+/tcdA+/tcdC+/cdd3+ profile was the predominant pattern and cdtA and cdtB genes were found in one-third of the isolates. Using the SlpA genotyping, 12 main genotypes and 16 subtypes were identified. The SlpA type 078-1 was the most prevalent genotype (20.8%), and identified within the isolates of RT126. The yok-1, gr-1, cr-1 and kr-3 genotypes were detected in 14.5%, 12.5%, 12.5% and 8.3% of isolates, respectively. Almost all the isolates with the same RT were clustered in similar SlpA sequence types. In comparison to PCR ribotyping, SlpAST, as a simple and highly reproducible sequenced-based technique, can discriminate well between C. difficile isolates. This typing method appears to be a valuable tool for the epidemiological study of C. difficile isolates worldwide.

Molecular characterization of pathogenicity locus (PaLoc) and tcdC genetic diversity among tcdA+B+Clostridioides difficile clinical isolates in Tehran, Iran.

Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs). Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene. Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene. The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/toxinotype 0 and the RT 126/toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

Coexistence of Clostridioides difficile and Staphylococcus aureus in gut of Iranian outpatients with underlying inflammatory bowel disease.

Clostridioides difficile and Staphylococcus aureus are two well-known pathogens both causing hospital- and community-acquired infections. However, their intestinal coexistence was not well investigated in inflammatory bowel disease (IBD). Herein, we explored the prevalence of C. difficile, S. aureus and their coexistence in the gut of Iranian patients with IBD. Fecal and colon specimens were obtained from 70 outpatients with underlying IBD, and investigated for the presence of C. difficile and S. aureus. C. difficile isolates were characterised by CE-ribotyping. PCR was used for detection of toxin-encoding genes of C. difficile and S. aureus isolates. The antimicrobial susceptibility testing of C. difficile and S. aureus isolates were examined by agar dilution and Kirby-Bauer disk diffusion methods, respectively. Totally, C. difficile and S. aureus were detected in only 5.7% and 15.8% of IBD flares. Coexistence of C. difficile and S. aureus was detected in 5.7% of IBD flares. Two different C. difficile ribotypes including RT 126 and RT 017 were identified showing toxin profiles of tcdA+B+/cdtA+B+ and tcdA+B+, respectively. In S. aureus isolates, only positivity for the presence of sea enterotoxin was detected. C. difficile isolates were susceptible to metronidazole, ceftazidime and fidaxomicin. The highest resistance of S. aureus isolates was observed against penicillin (92.3%), following amoxicillin-clavulanate (38.5%) and amikacin (30.8%). Our findings demonstrated that patients with IBD flare are more sensitive to acquire coinfection of C. difficile and S. aureus than remission. However, more robust data is required to study the crosstalk between these enteric infections and their clinical relevance in patients with IBD flare.

Immunomodulation and Generation of Tolerogenic Dendritic Cells by Probiotic Bacteria in Patients with Inflammatory Bowel Disease.

In inflammatory bowel diseases (IBD), the therapeutic benefit and mucosal healing from specific probiotics may relate to the modulation of dendritic cells (DCs). Herein, we assessed the immunomodulatory effects of four probiotic strains including Lactobacillus salivarius, Bifidobacterium bifidum, Bacillus coagulans and Bacillus subtilis natto on the expression of co-stimulatory molecules, cytokine production and gene expression of signal-transducing receptors in DCs from IBD patients. Human monocyte-derived DCs from IBD patients and healthy controls were exposed to four probiotic strains. The expression of co-stimulatory molecules was assessed and supernatants were analyzed for anti-inflammatory cytokines. The gene expression of toll-like receptors (TLRs), IL-12p40 and integrin αvß8 were also analyzed. CD80 and CD86 were induced by most probiotic strains in ulcerative colitis (UC) patients whereas only B. bifidum induced CD80 and CD86 expression in Crohn's disease (CD) patients. IL-10 and TGF-ß production was increased in a dose-independent manner while TLR expression was decreased by all probiotic bacteria except B. bifidum in DCs from UC patients. TLR-4 and TLR-9 expression was significantly downregulated while integrin ß8 was significantly increased in the DCs from CD patients. IL-12p40 expression was only significantly downregulated in DCs from CD patients. Our findings point to the general beneficial effects of probiotics in DC immunomodulation and indicate that probiotic bacteria favorably modulate the expression of co-stimulatory molecules, proinflammatory cytokines and TLRs in DCs from IBD patients.

Detection of B. fragilis group and diversity of bft enterotoxin and antibiotic resistance markers cepA, cfiA and nim among intestinal Bacteroides fragilis strains in patients with inflammatory bowel disease.

We compared frequency of the members of B. fragilis group in 100 and 20 colon biopsy specimens of inflammatory bowel disease (IBD) and non-IBD patients. Agar dilution and PCR were orderly used to detect minimal inhibitory concentration of ampicillin, imipenem, and metronidazole, and carriage of related resistance genes cepA, cfi, and nim. B. fragilis group was detected in 38% of IBD (UC: 36/89; CD:1/11) and 25% (5/20) of non-IBD patients. While B. vulgatus (UC: 20/36, CD: 1/2, control: 1/6); B. fragilis (UC: 18/36, CD: 1/2, control: 5/6); B. ovatus (UC: 2/36); B. caccae (UC: 1/36); and B. eggerthii (UC: 1/36) were characterized, colonization of B. thetaiotamicron, B. merdae, B. distasonis, B. stercoris and B. dorei species was not detected in these specimens. Co-existence of B. fragilis + B. vulgatus (5 patients) and B. vulgatus + B. caccae (1 patient) was detected just in UC patients. bft was detected among 31.5% (6/19) of B. fragilis strains in the IBD and 40% (2/5) in the non-IBD groups. Nearly, 73.6% of the strains from the patient group and 80% in control group harbored cepA; 31.5% and 20% in the patients and control groups harbored cfiA, and none of them harbored nim determinant. Co-occurrence of the cepA and cfiA was orderly detected in 10.5% (2/19) and 20% (1/5) of the strains in these groups. The resistance rates were detected as 95.8% (23/24 (to ampicillin (MIC range of ≤0.5-≥16 µg/ml), 0% to metronidazole and 29.1% to imipenem (7/24, MIC range ≤4-32 µg/ml). Nearly 25% (6/24) of the strains were resistant to ampicillin and imipenem, simultaneously. No statistically significant difference was detected between the IBD and control groups for drug resistance phenotypes. Statistical analysis showed significant associations between resistance to ampicillin or imipenem and carriage of cepA or cfiA, respectively (p value = 0.0007). PCR results on the extracted plasmids confirmed their roles in carriage of cfiA and cepA. These data provide guide for antibiotic therapy and highlights wide distribution of ß-lactam resistant B. fragilis strains in patients with IBD and non-IBD intestinal disorders.

Molecular typing of Clostridium difficile isolates cultured from patient stool samples and gastroenterological medical devices in a single Iranian hospital.

This study aimed to characterize Clostridium difficile isolates cultured from stool samples of patients with C. difficile infection (CDI) and swabs from a medical environment in a gastroenterology center in Tehran, Iran. A total of 158 samples (105 stool samples from hospitalized patients and 53 swabs from medical devices and the environment) were collected from January 2011 to August 2011 and investigated for the presence of C. difficile by direct anaerobic culture on a selective media for C. difficile. C. difficile isolates were further characterized by capillary electrophoresis (CE) ribotyping and toxin gene multiplex PCR. Of 158 samples, C. difficile was cultured in 19 of 105 stool samples (18%) and in 4 of 53 swabs (7.5%). C. difficile PCR ribotype (RT) 126 was the most common RT in the study (21.7%). Further RTs were: 001, 003, 014, 017, 029, 039, 081, 103 and 150. RTs 126, 001, 150 were cultured from both the stool samples and swabs of medical devices and the hospital environment which suggest a possible route of transmission.

The frequency of bacterial contamination and diversity of drug resistance patterns in devices and staff of endoscopy and colonoscopy units.

Background: This study aimed at analyzing microbial contamination in medical equipment, environment, and staff of a gastroenterology unit. Methods: Samples of gastrointestinal imaging devices, the environment, and staff were collected using standard swab-rinse technique and biochemical or molecular characteristics of the isolates, their susceptibility to antibiotics, and similarity of the resistance patterns were investigated. Results: Out of 107 samples, bacterial contamination was detected in the hands of staff (54.1%), imaging devices (56.7%), and in the environment (54.5%). While Pseudomonas spp. were detected only in the imaging devices (13.5%), Bacillus spp. (32.4% and 31.5%), Enterococcus spp. (14.3% and 5.9%), Clostridium difficile (10.8% and 10.5%), and Staphylococcus epidermidis (5.4% and 15.9%) were orderly the most common isolates from samples of the imaging devices and the environment. Nearly, 40% of P. aeruginosa strains were resistant to cefepime, while resistance to cephalosporins and ß-lactamase inhibitor was detected in 33% and 75% of S. aureus strains, respectively. Homology of resistance patterns was detected between the imaging devices and hands of the staff. Conclusion: Our results proposed biofilm and spore forming bacteria as main contaminants of imaging devices in this hospital. Homology of the resistance patterns proposed involvement of staff in contamination of the equipment.

An ultrasensitive genosensor for detection of toxigenic and non-toxigenic Clostridioides difficile based on a conserved sequence in surface layer protein coding gene.

Clostridioides difficile (C. difficile) is the most common agent of antibiotic-associated diarrhea, leading to intestinal infection through the secretion of two major toxins. Not all strains of this bacterium are toxigenic, but some of them cause infection via their accessory virulence factors, such as surface layer protein (SlpA). SlpA is conserved in both toxigenic and non-toxigenic strains of C. difficile. In the present work, an amplification-free electrochemical genosensor was designed for the detection of the slpA gene. A glassy carbon electrode coated with gold nanoparticle-reduced graphene oxide nanocomposite was used as the working electrode, and its surface was modified using a simple thiolated linear oligonucleotide as the bioreceptor. Moreover, the hexaferrocenium tri[hexa(isothiocyanato) iron(III)] trihydroxonium (HxFc) complex was used as an intercalator, and its redox signal was recorded using differential pulse voltammetry. Scan rate studies indicated a quasi-reversible adsorption-controlled process for the HxFc complex. This genosensor showed high sensitivity with a limit of detection of 0.2 fM, a linear response range of 0.46-1900 fM, and a satisfactory specificity toward the synthetic slpA target gene. Also, the genosensor indicated responses in the mentioned linear range toward the genome extracted from either toxigenic or non-toxigenic strains of C. difficile.