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Clostridioides difficile is the leading cause of worldwide antibiotics-associated diarrhea. In this study, we report the construction and evaluation of a novel bacteriophage lysin-human defensin fusion protein targeting C. difficile. The fusion protein, designated LHD, is composed of two parts connected by a 3-repeating unit linker “(GGGGS)3”: the catalytic domain of a lysin protein from a C. difficile bacteriophage phiC2 (LCD), and the functional domain of a human defensin protein HD5. Lytic assays showed that LHD protein had a potent lytic activity against different types of clinical C. difficile strains, including the epidemic 027, 078, 012, and 087 strains. The minimum inhibitory concentration (MIC) of LHD was 0.78 μg/ml, which was lower than the MIC of the protein LCD (1.56 μg/ml), and the MICs of metronidazole (4 μg/ml) and vancomycin (4 μg/ml). In addition, the LHD protein could lyse C. different strains in different pHs (6.0, 7.0, and 8.0). Evaluation of LHD potency in vivo using mouse model of C. difficile infection (CDI) showed that administration of the LHD protein (twice daily for 7 days) was effective in mitigating the symptoms and reducing the death from CDI. Treatment with LHD also significantly decreased the number of C. difficile spores and the toxin level in feces from the infected mice. Our data suggest that this novel lysin-human defensin fusion protein has a potential on CDI control.

Weaning is one of the most stressful challenges in the pig’s life, which contributes to dysfunctions of intestinal and immune system, disrupts the gut microbial ecosystem, and therefore compromises the growth performance and health of piglets. To mitigate the negative impact of the stress on early-weaned piglets, effective measures are needed to promote gut health. Toward this end, we tamed a Saccharomyces cerevisiae strain and developed a probiotic Duan-Nai-An, which is a yeast culture of the tamed S. cerevisiae on egg white. In this study, we tested the performance of Duan-Nai-An on growth and health of early-weaned piglets and analyzed its impact on fecal microbiota. The results showed that Duan-Nai-An significantly improved weight gain and feed intake, and reduced diarrhea and death of early-weaned piglets. Analysis of the gut microbiota showed that the bacterial community was shaped by Duan-Nai-An and maintained as a relatively stable structure, represented by a higher core OTU number and lower unweighted UniFrac distances across the early weaned period. However, fungal community was not significantly shaped by the yeast probiotics. Notably, 13 bacterial genera were found to be associated with Duan-Nai-An feeding, including Enterococcus, Succinivibrio, Ruminococcus, Sharpea, Desulfovibrio, RFN20, Sphaerochaeta, Peptococcus, Anaeroplasma, and four other undefined genera. These findings suggest that Duan-Nai-An has the potential to be used as a feed supplement in swine production.

The global prevalence of drug resistance has created an urgent need for the discovery of novel anti-infective drugs. The major source of antibiotics in current clinical practice is terrestrial actinobacteria; the less-exploited deep-sea actinobacteria may serve as an unprecedented source of novel natural products. In this study, we evaluated 50 actinobacteria strains derived from diverse deep water sponges and environmental niches for their anti-microbial activities against a panel of pathogens including Candida albicans, Clostridium difficile, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa. More than half of the tested strains (27) were identified as active in at least one assay. The rare earth salt lanthanum chloride (LaCl3) was shown to be as an effective elicitor. Among the 27 strains, the anti-microbial activity of 15 were induced or enhanced by the addition of LaCl3. This part of study focused on one strain R818, in which potent antifungal activity was induced by the addition of LaCl3. We found that the LaCl3-activated metabolites in R818 are likely antimycin-type compounds. One of them, compound 1, has been purified. Spectroscopic analyses including HR-MS and 1D NMR indicated that this compound is urauchimycin D. The antifungal activity of compound 1 was confirmed with a minimal inhibitory concentration (MIC) of 25 μg/mL; the purified compound also showed a moderate activity against C. difficile. Additional notable strains are: strain N217 which showed both antifungal and antibacterial (including P. aeruginosa) activities and strain M864 which showed potent activity against C. difficile with an MIC value (0.125 μg/mL) lower than those of vancomycin and metronidazole. Our preliminary studies show that deep-sea actinobacteria is a promising source of anti-infective natural products.

Background:Clostridioides difficile infection (CDI) is an important cause of morbidity and mortality among hospitalized patients. In China, however, hospital staff do not routinely test for CDI, leading to under-diagnosis and poor patient outcomes. Locally generated CDI data can help assess the magnitude of the problem and strengthen approaches for CDI prevention and control.Methods: We prospectively monitored hospital-onset hospital-associated (HOHA) CDI in four intensive care units (ICUs) from June 2013 to September 2014 in a large teaching hospital in China. We collected clinical information from all ICU patients with ≥ 3 episodes of diarrhea occurring within a 24-h period at least 48 h following admission (suspect case definition). Stool specimens were collected from all suspect cases of CDI and cultured for C. difficile. Polymerase chain reaction (PCR) was used to detect toxin genes from positive isolates; multi-locus sequence typing (MLST) was used for typing and identifying novel strains. We estimated the incidence rate as the number of HOHA CDI cases per 10,000 patient days; 95% confidence intervals were generated to assess rate differences between the four ICUs.Results: A total of 593 hospital-onset diarrhea patients met the suspect case definition during the study period. Of these, 47 patients (8%) were positive for C. difficile and toxin genes. The HOHA-CDI incidence rate was 14.1 cases per 10,000 patient days (95% CI: 10.5–18.6). Six patients with HOHA CDI died. ST54 (n = 14, 20%) was the most common type of HOHA-CDI strain circulating in the hospital during the study period and was linked to a temporal cluster (outbreak) involving two (NICU and GICU) of the four ICUs.Conclusion: HOHA-CDI occurs among ICU patients at this teaching hospital, supporting the importance of routine testing for CDI. Information on strain distribution can help detect CDI outbreaks. Detection of ST54 strain in a temporal cluster suggests possible gaps in infection control practices that should be investigated and addressed as needed.

PurposeClostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes.MethodsRecently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a.ResultsThe platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/μL and 1 copy/μL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection.ConclusionIn conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.