The complete catalog of antimicrobial resistance secondary active transporters in Clostridioides difficile: evolution and drug resistance perspective.

Secondary active transporters shuttle substrates across eukaryotic and prokaryotic membranes, utilizing different electrochemical gradients. They are recognized as one of the antimicrobial efflux pumps among pathogens. While primary active transporters within the genome of C. difficile 630 have been completely cataloged, the systematical study of secondary active transporters remains incomplete. Here, we not only identify secondary active transporters but also disclose their evolution and role in drug resistance in C. difficile 630. Our analysis reveals that C. difficile 630 carries 147 secondary active transporters belonging to 27 (super)families. Notably, 50 (34%) of them potentially contribute to antimicrobial resistance (AMR). AMR-secondary active transporters are structurally classified into five (super)families: the p-aminobenzoyl-glutamate transporter (AbgT), drug/metabolite transporter (DMT) superfamily, major facilitator (MFS) superfamily, multidrug and toxic compound extrusion (MATE) family, and resistance-nodulation-division (RND) family. Surprisingly, complete RND genes found in C. difficile 630 are likely an evolutionary leftover from the common ancestor with the diderm. Through protein structure comparisons, we have potentially identified six novel AMR-secondary active transporters from DMT, MATE, and MFS (super)families. Pangenome analysis revealed that half of the AMR-secondary transporters are accessory genes, which indicates an important role in adaptive AMR function rather than innate physiological homeostasis. Gene expression profile firmly supports their ability to respond to a wide spectrum of antibiotics. Our findings highlight the evolution of AMR-secondary active transporters and their integral role in antibiotic responses. This marks AMR-secondary active transporters as interesting therapeutic targets to synergize with other antibiotic activity.

Two Recombinant Bacteriocins, Rhamnosin and Lysostaphin, Show Synergistic Anticancer Activity Against Gemcitabine-Resistant Cholangiocarcinoma Cell Lines.

Cholangiocarcinoma (CCA), a bile duct cancer with a high mortality rate, has a poor prognosis due to its highly invasive and drug-resistant phenotypes. More effective and selective therapies are urgently needed. Bacteriocins are broad-spectrum antimicrobial peptides/proteins produced by bacterial strains to compete with other bacteria. Recent studies have reported that bacteriocins exhibit anticancer properties against various cancer cell lines with minimal toxicity toward normal cells. In this study, two types of recombinant bacteriocins, rhamnosin from probiotic Lacticaseibacillus rhamnosus and lysostaphin from Staphylococcus simulans, were highly produced in Escherichia coli and subsequently purified via immobilized-Ni2+ affinity chromatography. When their anticancer activity was investigated against CCA cell lines, both rhamnosin and lysostaphin were found capable of inhibiting the growth of CCA cell lines in a dose-dependent fashion but were less toxic toward a normal cholangiocyte cell line. Rhamnosin and lysostaphin as single treatments could suppress the growth of gemcitabine-resistant cell lines to the same extent as or more than they suppressed the parental counterparts. A combination of both bacteriocins more strongly inhibited growth and enhanced cell apoptosis in both parental and gemcitabine-resistant cells partly through the increased expression of the proapoptotic genes BAX, and caspase-3, -8, and -9. In conclusion, this is the first report to demonstrate an anticancer property of rhamnosin and lysostaphin. Using these bacteriocins as single agents or in combination would be effective against drug-resistant CCA.

Enterococcus faecalis Isolated From Infant Feces Inhibits Toxigenic Clostridioides (Clostridium) difficile.

Clostridioides (Clostridium) difficile infection is implicated as a major cause of antibiotic-associated diarrhea in hospitals worldwide. Probiotics, especially lactic acid bacteria, are the most frequently used alternative treatment. This study aims to identify potential probiotic enterococci strains that act against C. difficile strains and exert a protective effect on colon adenocarcinoma cells (HT-29 cells). To this end, nine Enterococcus strains isolated from the feces of breast-fed infants were investigated. They were identified as E. faecalis by 16s rRNA sequencing and MALDI-TOF. The probiotic properties including their viabilities in simulated gastrointestinal condition, cell adhesion ability, and their safety were evaluated. All strains exhibited more tolerance toward both pepsin and bile salts and adhered more tightly to HT-29 cells compared with the reference probiotic strain Lactobacillus plantarum ATCC 14917. Polymerase chain reaction (PCR) results exhibited that six of nine strains carried at least one virulence determinant gene; however, none exhibited virulence phenotypes or carried transferable antibiotic resistance genes. These strains did not infect Galleria mellonella when compared to pathogenic E. faecalis strain (p < 0.05). Moreover, their antibacterial activities against C. difficile were examined using agar well-diffusion, spore production, and germination tests. The six safe strains inhibited spore germination (100 - 98.20% ± 2.17%) and sporulation, particularly in C. difficile ATCC 630 treated with E. faecalis PK 1302. Furthermore, immunofluorescence assay showed that the cytopathic effects of C. difficile of HT-29 cells were reduced by the treatment with the cell-free supernatant of E. faecalis strains. These strains prevented rounding of HT-29 cells and preserved the F-actin microstructure and tight junctions between adjacent cells, which indicated their ability to reduce the clostridial cytopathic effects. Thus, the study identified six E. faecalis isolates that have anti-C. difficile activity. These could be promising probiotics with potential applications in the prevention of C. difficile colonization and treatment of C. difficile infection.

Role of the Sea Urchin Stomopneustes variolaris (Lamarck, 1816) Pits as a Habitat for Epilithic Macroinvertebrates on a Tropical Intertidal Rocky Shore.

Many sea urchin species excavate pits in sedimentary rock, transforming primary rocky substrates into sea urchins' pits. These pits are not only used as their home but seem to harbor a distinct assemblage of organisms. We investigated small-scale spatial variation in community of macroinvertebrates by comparing community composition of epilithic macroinvertebrates between those found on unmodified rocky substrate, inside pits occupied by rock-boring sea urchin Stomopneustes variolaris (Lamarck, 1816), and unoccupied pits, on an intertidal rocky shore in southern Thailand. Size structures of macroinvertebrates were compared between pits and analyses were performed to investigate whether the use of habitat depends on availability of space, or biological interactions between sea urchins and other macroinvertebrates. Size structure of the most abundant mobile fauna, top shells Trochus radiatus Gmelin, 1791, were also analyzed to assess whether they exhibit ontogenetic changes in habitat use. Although a few species were found in all habitat types, community compositions were different. Chitons and limpets were found exclusively on unmodified substrate; whereas relatively large-sized gastropods inhabited unoccupied pits, and occupied pits harbored small-sized crustacean and gastropod species. Generally, in occupied pits, small-sized faunas were more abundant than larger faunas, suggesting that sea urchin's body may function as a biogenic structure providing refugia for small-sized individuals. In unoccupied pits volume of all macroinvertebrates increased as available space increased. This was not observed in occupied pits, where disturbances due to sea urchin's activities may be more important in determining habitat use.