Development of an efficient antimicrobial susceptibility testing method with species identification by Nanopore sequencing of 16S rRNA amplicons.

While amplicon sequencing of 16S rRNA is a common method for studying microbial community, it has been difficult to identify genera and species using next-generation sequencers to examine some regions (e.g., V3-V4 of 16S rRNA) because of the short read lengths. However, the advent of third-generation sequencers has made it possible to analyze the full length of the 16S rRNA gene, which allowed for species level identification at low cost. In this study, we evaluated the accuracy of the identification with a third-generation sequencer, MinION from Oxford Nanopore Technologies, using nine indigenous bacteria that can pose problems with food poisoning and opportunistic infections as an example. We demonstrated that Enterococcus faecalis and Enterococcus hirae could be identified at the species level with an accuracy of 96.4% to 97.5%. We also demonstrated that the absolute counts of various bacteria could be determined by spiking the sample with a bacterium as an internal standard. Then, we tested whether this convenient bacterial identification method could evaluate the antibiotic sensitivities of multiple bacteria simultaneously. In order to evaluate antimicrobial susceptibility, a mock community, an artificial mixture of the nine bacterial strains, was prepared and cultured in the presence of the antibiotics ofloxacin or chloramphenicol, and the 16S rRNAs were analyzed by using Nanopore sequencer. We confirmed that antibiotic-induced cell count reductions could be measured simultaneously by quantifying the abundances of various bacteria in the mock community before and after culture. It was thus shown that the antibiotic sensitivities of multiple bacteria could be evaluated simultaneously, with distinction made between bactericidal action and bacteriostatic action. This methodology would allow rapid evaluation of antibiotic activity spectrum at the species level containing a wide variety of bacteria, such as biofilm bacteria and gut microbiota.

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle.

Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsXEG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

AldB controls persister formation in Escherichia coli depending on environmental stress.

Persisters are multidrug-tolerant cells that are present within antibiotic-sensitive populations. Persister formation is not induced by genetic mutations, but rather by changes in the degree of expression of some genes. High redundancy has been observed among the pathways that have been hypothesized to respond to specific stresses. In this study, we conducted RNA sequencing of Escherichia coli persisters under various stress conditions to identify common mechanisms. We induced stresses such as glucose or amino acid exhaustion, acid stress and anaerobic conditions, all of which are encountered during bacterial pathogenesis. We found that most genes are differentially expressed depending on the specific stress condition; however, some genes were commonly expressed in persisters in most stress conditions. Commonly expressed genes are expected to be promising therapeutic targets for combating persistent infections. We found that knockdown of aldehyde dehydrogenase (aldB), which was expressed in every condition except for acid stress, decreased persisters in the non-stressed condition. However, the same strain unexpectedly showed an increased number of persisters in the amino acid-limited condition. Because the increase in persister number is glycolytic metabolite-dependent, metabolic flow may play a crucial role in aldB-mediated persister formation. These data suggest that environmental stresses alter persister mechanisms. Identification of environmental influences on persister formation during pathogenesis is therefore necessary to enabling persister eradication.

Stochastic expression of lactate dehydrogenase A induces Escherichia coli persister formation.

Bacterial persisters are phenotypic variants that survive the treatment of lethal doses of growth-targeting antibiotics without mutations. Although the mechanism underlying persister formation has been studied for decades, how the persister phenotype is switched on and protects itself from antibiotics has been elusive. In this study, we focused on the lactate dehydrogenase gene (ldhA) that was upregulated in an Escherichia coli persister-enriched population. A survival rate assay using an ldhA-overexpressing strain showed that ldhA expression induced persister formation. To identify ldhA-mediated persister formation at the single-cell level, time-lapse microscopy with a microfluidic device was used. Stochastic ldhA expression was found to induce dormancy and tolerance against high-dose ampicillin treatment (500 µg/ml). To better understand the underlying mechanism, we investigated the relationship between ldhA-mediated persister formation and previously reported persister formation through aerobic metabolism repression. As a result, ldhA expression enhanced the proton motive force (PMF) and ATP synthesis. These findings suggest that ldhA-mediated persister formation pathway is different from previously reported persister formation via repression of aerobic metabolism.

Unique transcriptional profile of native persisters in Escherichia coli.

Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.

Thermophilic ethanol fermentation from lignocellulose hydrolysate by genetically engineered Moorella thermoacetica.

A transformant of Moorella thermoacetica was constructed for thermophilic ethanol production from lignocellulosic biomass by deleting two phosphotransacetylase genes, pdul1 and pdul2, and introducing the native aldehyde dehydrogenase gene (aldh) controlled by the promoter from glyceraldehyde-3-phosphate dehydrogenase. The transformant showed tolerance to 540mM and fermented sugars including fructose, glucose, galactose and xylose to mainly ethanol. In a mixed-sugar medium of glucose and xylose, all of the sugars were consumed to produce ethanol at the yield of 1.9mol/mol-sugar. The transformant successfully fermented sugars in hydrolysate prepared through the acid hydrolysis of lignocellulose to ethanol, suggesting that this transformant can be used to ferment the sugars in lignocellulosic biomass for ethanol production.

A water splitting system using an organo-photocathode and titanium dioxide photoanode capable of bias-free H2 and O2 evolution.

This study examined a water-splitting system comprising a TiO2 photoanode and an organo-photocathode consisting of a p-n bilayer. Stoichiometric decomposition of water into H2 and O2 successfully occurred at bias voltages lower than the theoretical value (i.e. 1.23 V). Compared to the conventional TiO2 and Pt systems, the proposed water-splitting system demonstrated water splitting without any externally applied bias.

Detection of pre-mRNA splicing in vitro by an RNA-templated fluorogenic reaction.

RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken δ-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process.