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.

Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil.

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC-MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the ß-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Frontiers in antibiotic alternatives for Clostridioides difficile infection.

Clostridioides difficile (C. difficile) is a gram-positive, anaerobic spore-forming bacterium and a major cause of antibiotic-associated diarrhea. Humans are naturally resistant to C. difficile infection (CDI) owing to the protection provided by healthy gut microbiota. When the gut microbiota is disturbed, C. difficile can colonize, produce toxins, and manifest clinical symptoms, ranging from asymptomatic diarrhea and colitis to death. Despite the steady-if not rising-prevalence of CDI, it will certainly become more problematic in a world of antibiotic overuse and the post-antibiotic era. C. difficile is naturally resistant to most of the currently used antibiotics as it uses multiple resistance mechanisms. Therefore, current CDI treatment regimens are extremely limited to only a few antibiotics, which include vancomycin, fidaxomicin, and metronidazole. Therefore, one of the main challenges experienced by the scientific community is the development of alternative approaches to control and treat CDI. In this Frontier article, we collectively summarize recent advances in alternative treatment approaches for CDI. Over the past few years, several studies have reported on natural product-derived compounds, drug repurposing, high-throughput library screening, phage therapy, and fecal microbiota transplantation. We also include an update on vaccine development, pre- and pro-biotics for CDI, and toxin antidote approaches. These measures tackle CDI at every stage of disease pathology via multiple mechanisms. We also discuss the gaps and concerns in these developments. The next epidemic of CDI is not a matter of if but a matter of when. Therefore, being well-equipped with a collection of alternative therapeutics is necessary and should be prioritized.

Teicoplanin Suppresses Vegetative Clostridioides difficile and Spore Outgrowth.

In recent decades, the incidence of Clostridioides difficile infection (CDI) has remained high in both community and health-care settings. With the increasing rate of treatment failures and its ability to form spores, an alternative treatment for CDI has become a global priority. We used the microdilution assay to determine minimal inhibitory concentrations (MICs) of vancomycin and teicoplanin against 30 distinct C. difficile strains isolated from various host origins. We also examined the effect of drugs on spore germination and outgrowth by following the development of OD600. Finally, we confirmed the spore germination and cell stages by microscopy. We showed that teicoplanin exhibited lower MICs compared to vancomycin in all tested isolates. MICs of teicoplanin ranged from 0.03-0.25 µg/mL, while vancomycin ranged from 0.5-4 µg/mL. Exposure of C. difficile spores to broth supplemented with various concentrations of antimicrobial agents did not affect the initiation of germination, but the outgrowth to vegetative cells was inhibited by all test compounds. This finding was concordant with aberrant vegetative cells after antibiotic treatment observed by light microscopy. This work highlights the efficiency of teicoplanin for treatment of C. difficile through prevention of vegetative cell outgrowth.

Temporal Variations in Patterns of Clostridioides difficile Strain Diversity and Antibiotic Resistance in Thailand.

Clostridioides difficile has been recognized as a life-threatening pathogen that causes enteric diseases, including antibiotic-associated diarrhea and pseudomembranous colitis. The severity of C. difficile infection (CDI) correlates with toxin production and antibiotic resistance of C. difficile. In Thailand, the data addressing ribotypes, toxigenic, and antimicrobial susceptibility profiles of this pathogen are scarce and some of these data sets are limited. In this study, two groups of C. difficile isolates in Thailand, including 50 isolates collected from 2006 to 2009 (THA group) and 26 isolates collected from 2010 to 2012 (THB group), were compared for toxin genes and ribotyping profiles. The production of toxins A and B were determined on the basis of toxin gene profiles. In addition, minimum inhibitory concentration of eight antibiotics were examined for all 76 C. difficile isolates. The isolates of the THA group were categorized into 27 A-B+CDT- (54%) and 23 A-B-CDT- (46%), while the THB isolates were classified into five toxigenic profiles, including six A+B+CDT+ (23%), two A+B+CDT- (8%), five A-B+CDT+ (19%), seven A-B+CDT- (27%), and six A-B-CDT- (23%). By visually comparing them to the references, only five ribotypes were identified among THA isolates, while 15 ribotypes were identified within THB isolates. Ribotype 017 was the most common in both groups. Interestingly, 18 unknown ribotyping patterns were identified. Among eight tcdA-positive isolates, three isolates showed significantly greater levels of toxin A than the reference strain. The levels of toxin B in 3 of 47 tcdB-positive isolates were significantly higher than that of the reference strain. Based on the antimicrobial susceptibility test, metronidazole showed potent efficiency against most isolates in both groups. However, high MIC values of cefoxitin (MICs 256 µg/mL) and chloramphenicol (MICs ≥ 64 µg/mL) were observed with most of the isolates. The other five antibiotics exhibited diverse MIC values among two groups of isolates. This work provides evidence of temporal changes in both C. difficile strains and patterns of antimicrobial resistance in Thailand.

The repertoire of ABC proteins in Clostridioides difficile.

ATP-binding cassette (ABC) transporters belong to one of the largest membrane protein superfamilies, which function in translocating substrates across biological membranes using energy from ATP hydrolysis. Currently, the classification of ABC transporters in Clostridioides difficile is not complete. Therefore, the sequence-function relationship of all ABC proteins encoded within the C. difficile genome was analyzed. Identification of protein domains associated with the ABC system in the C. difficile 630 reference genome revealed 226 domains: 97 nucleotide-binding domains (NBDs), 98 transmembrane domains (TMDs), 30 substrate-binding domains (SBDs), and one domain with features of an adaptor protein. Gene organization and transcriptional unit analyses indicated the presence of 78 ABC systems comprising 28 importers and 50 exporters. Based on NBD sequence similarity, ABC transporters were classified into 12 sub-families according to their substrates. Interestingly, all ABC exporters, accounting for 64% of the total ABC systems, are involved in antibiotic resistance. Based on analysis of ABC proteins from 49 C. difficile strains, the majority of core NBDs are predicted to be involved in multidrug resistance systems, consistent with the ability of this organism to survive exposure to an array of antibiotics. Our findings herein provide another step toward a better understanding of the function and evolutionary relationships of ABC proteins in this pathogen.

Isolation and characterization of thermophilic cellulose and hemicellulose degrading bacterium, Thermoanaerobacterium sp. R63 from tropical dry deciduous forest soil.

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.

Repurposing a platelet aggregation inhibitor ticagrelor as an antimicrobial against Clostridioides difficile.

Drug resistance in Clostridioides difficile becomes a public health concern worldwide, especially as the hypervirulent strains show decreased susceptibility to the first-line antibiotics for C. difficile treatment. Therefore, the simultaneous discovery and development of new compounds to fight this pathogen are urgently needed. In order to determinate new drugs active against C. difficile, we identified ticagrelor, utilized for the prevention of thrombotic events, as exhibiting potent growth-inhibitory activity against C. difficile. Whole-cell growth inhibition assays were performed and compared to vancomycin and metronidazole, followed by determining time-kill kinetics against C. difficile. Activities against biofilm formation and spore germination were also evaluated. Leakage analyses and electron microscopy were applied to confirm the disruption of membrane structure. Finally, ticagrelor's ability to synergize with vancomycin and metronidazole was determined using checkerboard assays. Our data showed that ticagrelor exerted activity with a MIC range of 20-40 µg/mL against C. difficile. This compound also exhibited an inhibitory effect on biofilm formation and spore germination. Additionally, ticagrelor did not interact with vancomycin nor metronidazole. Our findings revealed for the first time that ticagrelor could be further developed as a new antimicrobial agent for fighting against C. difficile.

Antimicrobial Effect of Asiatic Acid Against Clostridium difficile Is Associated With Disruption of Membrane Permeability.

Antibiotic resistance is a major concern in Clostridium difficile, the causative agent of antibiotic-associated diarrhea. Reduced susceptibility to first- and second-line agents is widespread, therefore various attempts have been made to seek alternative preventive and therapeutic strategies against this pathogen. In this work, the antimicrobial properties of asiatic acid were evaluated against C. difficile. Asiatic acid displayed substantial inhibitory effects on 19 C. difficile isolates collected from different sources with minimal inhibitory concentrations ranging from 10 to 20 µg/ml. Time kill analysis and minimal bactericidal concentration revealed potential bactericidal activity of this compound. Asiatic acid induced membrane damages and alterations in morphological ultrastructure in C. difficile, thereby causing the leakage of intracellular substances. Moreover, asiatic acid also displayed an inhibitory effect on cell motility, but did not interfere with biofilm formation and spore germination. Analysis of drug combination showed no synergistic effect between asiatic acid and vancomycin/metronidazole. Altogether, asiatic acid exhibited strong antimicrobial activity against vegetative cells and could serve as an alternative resource for tackling C. difficile.

Microbial Diversity in Sediments from the Bottom of the Challenger Deep, the Mariana Trench.

The Challenger Deep is the deepest ocean on Earth. The present study investigated microbial community structures and geochemical cycles associated with the trench bottom sediments of the Challenger Deep, the Mariana Trench. The SSU rRNA gene communities found in trench bottom sediments were dominated by the bacteria Chloroflexi (SAR202 and other lineages), Bacteroidetes, Planctomycetes, "Ca. Marinimicrobia" (SAR406), and Gemmatimonadetes and by the archaeal α subgroup of MGI Thaumarchaeota and "Ca. Woesearchaeota" (Deep-sea Hydrothermal Vent Euryarchaeotic Group 6). The SSU rRNA gene sequencing analysis indicated that the dominant populations of the thaumarchaeal α group in hadal water and sediments were similar to each other at the species or genus level. In addition, the co-occurrence of nitrification and denitrification was revealed by the combination of pore water geochemical analyses and quantitative PCR for nitrifiers.

CD2068 potentially mediates multidrug efflux in Clostridium difficile.

Clostridium difficile is a major cause of antibiotic-associated diarrhea and the treatment thereof becomes more difficult owing to a rise of multidrug resistant strains. ATP-binding cassette (ABC) transporters are known to play a crucial role in the resistance to multiple antibiotics. In this study, the potential contribution of an ABC transporter in C. difficile multidrug resistance was investigated. The expression level of the cd2068 gene in C. difficile encoding an ABC transporter was up-regulated following the exposure to certain antibiotics compared to the control cells. Heterologous expression of CD2068 in Escherichia coli revealed that it mediated the efflux of fluorescent substrates and conferred resistance to multiple drugs. The CD2068-associated ATPase activity in membrane vesicles was also stimulated by various antibiotics. Furthermore, the insertional inactivation of the cd2068 gene in C. difficile led to a significant increase in susceptibility to antibiotics, which could be genetically complemented, supporting that CD2068 was directly associated to the drug resistance. These results demonstrate the potential role for the ABC transporter CD2068 in the resistance mechanism against multiple drugs in C. difficile.

Insights into drug resistance mechanisms in Clostridium difficile.

The incidence of Clostridium difficile infection has been elevated and becoming common in hospitals worldwide. Although antibiotics usually serve as the primary treatment for bacterial infection including C. difficile infection, limitations and failures have been evident due to drug resistance. Antibiotic resistance in C. difficile has been recognized as one of the most important factors to promote the infection and increase the level of severity and the recurrence rate. Several outbreaks in many countries have been linked to the emergence of hypervirulent drug-resistant strains. This pathogen harbours various mechanisms against the actions of antibiotics. The present study highlights three main drug-resistant strategies in C. difficile including drug inactivation, target modification and efflux pump. Other mechanisms that potentially contribute to drug-resistant traits in this organism are also discussed.

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis.

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S(0)) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H2S from S(0) and free CoA-SH was observed, and the addition of enzyme and NADPH enhanced H2S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA-SH acted to solubilize S(0) by forming CoA persulfides, followed by reduction of an enzyme-S-S-CoA intermediate produced after both enzymatic and non-enzymatic evolution of H2S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA-SH, implying a direct reaction with S(0). TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA-SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S(0)-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S(0), suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S(0) was absent.