Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant.

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.

Gut Microbiota-Gut Metabolites and Clostridioides difficile Infection: Approaching Sustainable Solutions for Therapy.

Clostridioides difficile (C. difficile) infection (CDI) is the most common hospital-acquired infection. With the combination of a high rate of antibiotic resistance and recurrence, it has proven to be a debilitating public health threat. Current treatments for CDI include antibiotics and fecal microbiota transplantation, which contribute to recurrent CDIs and potential risks. Therefore, there is an ongoing need to develop new preventative treatment strategies for CDI. Notably, gut microbiota dysbiosis is the primary risk factor for CDI and provides a promising target for developing novel CDI therapy approaches. Along with gut microbiota dysbiosis, a reduction in important gut metabolites like secondary bile acids and short-chain fatty acids (SCFAs) were also seen in patients suffering from CDI. In this review study, we investigated the roles and mechanisms of gut microbiota and gut microbiota-derived gut metabolites, especially secondary bile acids and SCFAs in CDI pathogenesis. Moreover, specific signatures of gut microbiota and gut metabolites, as well as different factors that can modulate the gut microbiota, were also discussed, indicating that gut microbiota modulators like probiotics and prebiotics can be a potential therapeutic strategy for CDI as they can help restore gut microbiota and produce gut metabolites necessary for a healthy gut. The understanding of the associations between gut microbiota-gut metabolites and CDI will allow for developing precise and sustainable approaches, distinct from antibiotics and fecal transplant, for mitigating CDI and other gut microbiota dysbiosis-related diseases.

Sequential application of soil washing and phytoremediation in the land of fires.

This paper presents an experimental study aimed at verifying the efficiency of a double-stage remediation process to be applied in former agricultural sites contaminated by illegal dumping of industrial wastes. The process, which includes an EDDS (Ethylenediamine-N,N'-disuccinic acid) enhanced washing, followed by a phytoremediation treatment, is applied at the lab scale for the remediation of a soil sampled in a territory known as Land of Fires (Italy) contaminated with Cu (∼400 mg kg-1) and Zn (∼250 mg kg-1). Phytoremediation is conducted using Lactuca sativa to verify, together with process efficiency, the potential risks due to metal accumulation in edible species. The results of the washing process show the possibility of removing the potential toxic metals from 44% to 77% for Cu and from 18% to 47% for Zn. The removal is well distributed among all soil fractions. There is almost no removal of other components which are fundamental for an agricultural soil. Results of the subsequent phytoremediation treatment indicate that both the contaminants and the residual EDDS/EDDS-chelates adsorbed into the soil generally negatively affect plant growth, reducing the number of germinated seeds up to 43%, and the shoot length up to 63%. Nonetheless, whenever the efficiency of the washing stage is high enough, no adverse effect is obtained on the plants. The efficiency of the phytoremediation stage mainly relies on leaf uptake, which accounts for up to 88% of the total removed Cu and up to 95% of the total removed Zn. Stabilization in the underground part of the plant is more contained because of the limited mass of the roots.

Assessment of metal pollution in the Lambro Creek (Italy).

This study assessed the effect of metal pollution in the Lambro Creek (Southern Italy). Water, sediment and biota were collected at six sampling sites (June) for metal concentration assessment (Cr, Cu, Ni, Pb and Zn). Sequential extraction was performed to determine the distribution of metals in different geochemical sediment fractions. The influence of pH and leaching time on the release of metals from sediment to the water column was investigated via remobilization tests. A battery of toxicity tests (Vibrio fischeri, Raphidocelis subcapitata, Phaeodactylum tricornutum, and Daphnia magna) with multi-endpoints (bioluminescence, growth inhibition, and immobilization) was used to determine the overall toxicity in sediment water extracts. The results showed that metals did not exceed the probable effect concentration levels, with Cr concentration exceeding the threshold effect concentration level at all sampling points except for the one closer to the source of the creek, suggesting potential negative effect on the biota. Considering the cumulative criterion unit, sediment contamination was moderate at all sampling sites, except for L3 and L5 where biota was exposed to a very high risk. With respect to sequential analysis, the most readily available fraction of metal can be generalised as Ni > Cr > Cu > Zn > Pb. For better understanding the fate of metals in the water-sediment environment, their biogeochemical cycles should also be investigated in small creeks including both fresh (watercourse) and saltwater (river mouth) sediments.