Yuan-Pin Hung

Clostridioides difficile infection (CDI) poses a significant global health threat, exacerbated by the limitations of current antibiotic therapies, including high recurrence rates and severe gut microbiota dysbiosis. Here, we report the development of a novel bacteriocin-loaded DNA nanogel (NG) delivery system, engineered via genipin-crosslinking of genomic DNA extracted from kiwifruit, to address these clinical challenges. These CBMB/DNA NGs demonstrate exceptional structural stability and protective capacity within the harsh acidic environment in stomach, ensuring the safe delivery of the recombinant bacteriocin CBMB to the intestinal tract. Our findings reveal that CBMB/DNA NGs exhibit a robust pH-responsive release profile, preserving structural integrity during gastric transit while enabling controlled release in intestine. In a murine CDI model, the administration of CBMB/DNA NGs significantly enhances the therapeutic efficacy of vancomycin (VCM), leading to a marked reduction in CDI-associated clinical symptoms. Mechanistically, this combination therapy not only facilitates the efficient eradication of bacteria but also effectively promotes the rebalancing of gut microbiota. By combining the antimicrobial potency of bacteriocins with the biocompatibility of DNA-based nanocarriers, this study provides a promising, translatable strategy for improving the clinical management of CDI and offers new insights into leveraging biological polymers for targeted intestinal drug delivery.

Clostridioides difficile infection (CDI) remains a major healthcare challenge due to recurrent disease, spore persistence, and biofilm-associated tolerance, while conventional antibiotics often disrupt gut microbiota. Here, we report a star-shaped poly(l-lysine) dendrimer (G3-PLL₉) formulated into hyaluronic acid–based microgels for targeted oral delivery to the inflamed colon. G3-PLL₉ exhibited potent antimicrobial activity, including rapid bactericidal effects, superior spore inhibition compared with vancomycin, and robust biofilm disruption at subinhibitory concentrations. In a murine CDI model, rectal administration of G3-PLL₉ alleviated clinical symptoms, reduced tissue damage, and lowered recurrence risk. To enable oral therapy, G3-PLL₉ was incorporated into hyaluronic acid microgels, achieving site-specific release through hyaluronidase-mediated degradation in the inflamed colon. Importantly, treatment preserved commensal gut microbiota more effectively than vancomycin. Collectively, these findings highlight G3-PLL₉ microgels as a microbiota-sparing therapeutic that targets multiple stages of CDI pathogenesis─including spores and biofilms─and demonstrate their potential for clinical translation.

Asymptomatic Clostridioides difficile colonization (CdC) serves as a reservoir for pathogen transmission and may precede clinical infection. While risk factors for CdC have been well described in hospitalized populations, community-based data—particularly regarding lifestyle-associated factors and gut microbiota alterations—remain limited in Taiwan. This study aimed to determine the prevalence and risk factors of community-acquired CdC and to explore associated gut microbiota differences. We conducted a cross-sectional study analyzing 250 residual stool samples from adults aged ≥40 years who participated in community health screenings in Tainan City between 2006 and 2009. CdC was detected by polymerase chain reaction targeting the triosephosphate isomerase (tpi) gene. Demographic characteristics, lifestyle factors, and laboratory parameters were collected and analyzed. Exploratory 16S rRNA gene sequencing was performed on selected samples to compare gut microbiota composition between CdC and non-CdC groups. The prevalence of CdC was 11.6% (29/250). Cigarette smoking was identified as the sole independent factor associated with CdC (adjusted OR 2.35, 95% CI 1.05–5.28; P = 0.038). Exploratory microbiota analysis revealed differences in community composition between CdC and non-CdC samples, including increased relative abundance of Proteobacteria in CdC. Cigarette smoking was associated with an increased likelihood of community-acquired CdC in southern Taiwan. Distinct gut microbiota profiles were observed in individuals with CdC, supporting a potential link between smoking-related microbiota alterations and susceptibility to asymptomatic colonization. These findings underscore the relevance of modifiable lifestyle factors in the epidemiology of community CdC and warrant further investigation.

Clostridioides difficile infection is a global health concern, with increasing community-associated cases. Environmental sources may contribute to transmission. This study assessed hypervirulent C. difficile in wastewater and shellfish in southern Taiwan. From April to September 2020, water samples were collected from two domestic wastewater treatment plants (WWTPs), one hospital WWTP, and shellfish from an aquafarm in Tainan City. C. difficile isolates underwent molecular identification, toxin gene detection, ribotyping, antimicrobial susceptibility, and multi-locus variable-number tandem repeat analysis (MLVA). Ninety-seven C. difficile isolates representing 24 ribotypes were classified into 29 distinct strains—4, 13, and 12 from domestic WWTPs, hospital WWTP, and shellfish, respectively. Among 17 WWTP strains, 64.7% were toxigenic. Hypervirulent RT078 family strains (RT598, RT126, and RT127) with Δ39 base-pair deletion in toxin production control protein C gene were detected in WWTPs. One RT027 strain was discovered in clams—the first environmental report of RT027 in Taiwan. MLVA revealed this strain’s association with the American Type Culture Collection reference strain but distinct from other clinical strains. Five hypervirulent isolates harbored Thr82→Ile mutation in DNA gyrase subunit A, conferring high-level fluoroquinolone resistance (minimum inhibitory concentration >32 mg/L) while remaining susceptible to metronidazole and vancomycin. This first detection of hypervirulent RT078 in wastewater and RT027 in shellfish in Taiwan indicates that environmental reservoirs may contribute to community-associated infections, highlighting the need for expanded surveillance. IMPORTANCE: This study assessed the presence of Clostridioides difficile in wastewater and shellfish from southern Taiwan. Water samples were collected from both household and hospital treatment plants, along with clams from a local aquafarm. We identified several strains of C. difficile , including types specifically dangerous and resistant to certain antibiotics, such as fluoroquinolones. Notably, the hypervirulent RT027 strain, associated with severe infections in humans, was detected in shellfish, marking the first report of RT027 in environmental samples in Taiwan. Our findings indicate that food and water sources may contribute to the community transmission of C. difficile , highlighting the significance of ongoing environmental surveillance.

Objective: Clostridioides difficile is the most common cause of antibiotic-associated diarrhea. Wastewater from hospitals may be an important source of C. difficile transmission between hospitals and communities. The objective of this study is to quantify C. difficile spores and to elucidate their potential transmission risk via hospital wastewater. Methods: A prospective study of wastewater from a teaching hospital was conducted weekly, from July 2023 to June 2024. The number of C. difficile spores detected in wastewater from hospital settings fluctuated weekly during the study period. Results: There was a borderline association between the number of C. difficile spores detected in wastewater at room temperature in hospitals (p = 0.02) and the consumption of antimicrobial agents (p value = 0.04), particularly cephalosporins (p = 0.001). Specifically, the number of C. difficile spores detected in the wastewater was highly correlated with first-generation cephalosporin consumption (p = 0.002), particularly the consumption of first-generation intravenous cephalosporin (cefazolin) (p < 0.001). Conclusion: The number of C. difficile spores detected in wastewater from hospital settings is strongly associated with the consumption of antimicrobial agents, particularly cephalosporins. Further evaluation is needed to assess whether antibiotic stewardship programs can reduce the burden of C. difficile spores in wastewater. Plain Language Summary: Consumption of antimicrobial agents influences Clostridioides difficile spores in hospital wastewater.

Clostridioides difficile is an anaerobic, spore-forming pathogen responsible for illnesses ranging from mild diarrhea to life-threatening colitis. Current treatments rely on antibiotics such as vancomycin and metronidazole (MTZ), but high doses can disrupt gut microbiota, contributing to recurrent infections. Mitomycin C (MMC), a Food and Drug Administration-approved anticancer agent, is known to induce prophage activation in lysogenic bacteria. Given that over 70% of C. difficile strains harbor prophages, we evaluated MMC’s potential to enhance antibiotic efficacy against C. difficile infection (CDI). In vitro , MMC synergized with MTZ to inhibit strain R20291 and clinical isolates of RT027 and RT078 while reducing the minimum bactericidal concentration of MTZ against biofilm-associated cells. Ex vivo assays using mouse fecal suspensions confirmed the enhanced killing effect of the combination. In a murine recurrence model, low-dose MTZ + MMC treatment significantly improved survival and reduced fecal spore counts compared to monotherapies or vancomycin. Importantly, the combination did not cause greater liver or kidney toxicity than other antibiotics and resulted in less colonic epithelial damage. Microbiota profiling revealed that MTZ + MMC better preserved gut microbial composition than standard treatments. These findings suggest that low-dose MTZ combined with MMC enhances antimicrobial efficacy while reducing toxicity and microbiota disruption, offering a promising strategy for CDI management.

Introduction: Antimicrobial therapy plays a crucial role in the management of CDI patients. However, the standard agent for treating CDIs is limited to oral fidaxomicin or vancomycin. For patients made nil by mouth, there is a clinically urgent and essential need to develop an intravenous antibiotic.Methods: For C. difficile with the lowest MIC of nemonoxacin and vancomycin, the inhibitory effects were tested using the kinetic time-kill assay and ex vivo co-culture model. The effectiveness of nemonoxacin and vancomycin in inhibiting spore germination, the sporicidal activity, and the treatment of mice with CDIs were compared.Results: For clinical isolates and laboratory strains, lower MICs of nemonoxacin against C. difficile than levofloxacin and ciprofloxacin were observed, even in those harboring point mutations in the quinolone-resistance determining region. Although nemonoxacin failed to suppress spore outgrowth and germination in C. difficile, it exhibited an effective inhibitory effect against C. difficile in the kinetic time-kill assay and the ex vivo co-culture model. Mice receiving intraperitoneal nemonoxacin had less weight loss, higher cecum weight, a longer colon length, and lower expression of the tcdB gene, compared with untreated mice. Notably, there were no significant differences observed in weight loss, cecum weight, colon length, or tcdB gene expression between mice treated with vancomycin and those treated with any dose of nemonoxacin. Similarly, no significant differences were found between mice receiving combination therapy of intraperitoneal nemonoxacin plus oral vancomycin and those treated with intraperitoneal nemonoxacin or oral vancomycin alone.Discussion: The potential role of nemonoxacin, which can be administered parenterally, for treating CDIs was evidenced through the in vitro, ex vivo, and mouse models.