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.

Flumioxazin metabolism in pregnant animals and cell-based protoporphyrinogen IX oxidase (PPO) inhibition assay of fetal metabolites in various animal species to elucidate the mechanism of the rat-specific developmental toxicity.

Flumioxazin, an N-phenylimide herbicide, inhibits protoporphyrinogen oxidase (PPO), a key enzyme in heme biosynthesis in mammals, and causes rat-specific developmental toxicity. The mechanism has mainly been clarified, but no research has yet focused on the contribution of its metabolites. We therefore conducted in vivo metabolism studies in pregnant rats and rabbits, and found 6 major known metabolites in excreta. There was no major rat-specific metabolite. The most abundant component in rat fetuses was APF, followed by flumioxazin and 5 identified metabolites. The concentrations of flumioxazin and these metabolites in fetuses were lower in rabbits than in rats. In vitro PPO inhibition assays with rat and human liver mitochondria showed that flumioxazin is a more potent PPO inhibitor than the metabolites. There were no species differences in relative intensity of PPO inhibition among flumioxazin and these metabolites. Based on the results of these in vivo and in vitro experiments, we concluded that flumioxazin is the causal substance of the rat-specific developmental toxicity. As a more reliable test system for research on in vitro PPO inhibition, cell-based assays with rat, rabbit, monkey, and human hepatocytes were performed. The results were consistent with those of the mitochondrial assays, and rats were more sensitive to PPO inhibition by flumioxazin than humans, while rabbits and monkeys were almost insensitive. From these results, the species difference in the developmental toxicity was concluded to be due to the difference in sensitivity of PPO to flumioxazin, and rats were confirmed to be the most sensitive of these species.

Identification and in silico prediction of metabolites of the model compound, tebufenozide by human CYP3A4 and CYP2C19.

The metabolites of tebufenozide, a model compound, formed by the yeast-expressed human CYP3A4 and CYP2C19 were identified to clarify the substrate recognition mechanism of the human cytochrome P450 (CYP) isozymes. We then determined whether tebufenozide metabolites may be predicted in silico. Hydrogen abstraction energies were calculated with the density functional theory method B3LYP/6-31G(∗). A docking simulation was performed using FRED software. Several alkyl sites of tebufenozide were hydroxylated by CYP3A4 whereas only one site was modified by CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen abstraction energy estimation, respectively.

Metabolism of (Z)-(1R,3R)-Profluthrin in Rats.

When [benzyl-α-(14)C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6-8 h, and decreased (half-life; 37-52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the (14)C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ω-oxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions.

Metabolism of propyrisulfuron: 14C excretion, 14C concentration in plasma and tissues, and amount of metabolites in rats.

1. Metabolism of a novel sulfonylurea herbicide, propyrisulfuron [1-(2-chloro-6-propylimidazo[1,2-b]pyridazin-3-ylsulfonyl)-3-(4,6-dimethoxypyrimidin-2-yl)urea] labeled at the C-1 position of the propyl group and C-5 position of the pyrimidine ring with (14)C was investigated after a single oral administration in male and female rats. 2. Administered (14)C was excreted into the urine (5.7-29.8%) and feces (64.6-97.4%), respectively. (14)C concentration in plasma reached a maximum level at 4 to 12 h post-administration and then decreased rapidly with a biological half-life of approximately 23 to 32 h. Total (14)C residues in the whole body were <0.1-1.4%, suggesting that the residues were not accumulated in the tissues. 3. The amount of metabolites in urine, feces, and bile were quantified using high-performance liquid chromatography (HPLC). There were no differences in metabolites found between male and female rats. 4. The absorption for the low dose (5 mg/kg) and the high dose (1000 mg/kg) was estimated to be approximately 90% and 20%, respectively, suggesting a saturable absorption. 5. The plasma protein binding in male and female rats was ≥ 98.8%, suggesting that propyrisulfuron had a strong affinity to plasma proteins.

Identification of metabolites of propyrisulfuron in rats.

The metabolites found in the urine, feces and bile of male and female rats administered with (14)C-labeled herbicide, propyrisulfuron [1-(2-chloro-6-propylimidazo[1,2-b]pyridazin-3-ylsulfonyl)-3- (4,6-dimethoxypyrimidin-2-yl)urea] were identified by high-performance liquid chromatography (HPLC) with the ultraviolet (UV) and radioisotope (RI) detectors, tandem mass spectrometry and nuclear magnetic resonance (NMR). Administered (14)C was excreted into the urine (5.7-29.8%) and feces (64.6-97.4%). Urine and bile samples were concentrated and purified using a solid-phase extraction cartridge, and fecal homogenates were extracted using acetonitrile. Conjugates were hydrolyzed with enzyme or hydrochloric acid solution for identification. The proposed major metabolic reactions of propyrisulfuron are as follows: (1) hydroxylation of the pyrimidine ring, propyl group, and imidazopyridazine ring, (2) O-demethylation, (3) cleavage of the pyrimidine ring, and (4) glucuronic acid and sulfate conjugation. The metabolic patterns found are not different among sulfonylurea herbicides.

Metabolomic and transcriptomic profiling of human K-ras oncogene transgenic rats with pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.

Biotransformation and enzymatic reactions of synthetic pyrethroids in mammals.

Synthetic pyrethroids, a major insecticide group, are used worldwide for controlling indoor and agricultural pests. Extensive mammalian metabolism studies have been carried out since the late 1960s, and major metabolic reactions have been found to be oxidation of the acid or alcohol moiety, ester cleavage, and conjugation reactions. In addition, various conjugation reactions occur in mammals, forming hydrophilic and lipophilic conjugates. Pyrethroids are generally rapidly metabolized in mammals and completely excreted from the body in a short period. Human and laboratory animals share similar metabolic reactions for pyrethroids. Oxidation reactions in humans are mediated by several CYP isoforms. On the other hand, ester bonds of pyrethroids are hydrolyzed mainly by carboxylesterase(s).

In vitro metabolism of trans-permethrin and its major metabolites, PBalc and PBacid, in humans.

To estimate the metabolic profile of trans-permethrin in humans, a comparison of the in vitro metabolism of trans-permethrin in humans and rats was conducted using hepatic microsomes, and cytochrome P450 and UDP-glucuronyltransferase isoforms, which catalyze the metabolism of 3-phenoxybenzyl alcohol (PBalc) and 3-phenoxybenzoic acid (PBacid), respectively. In humans and rats, the major metabolic reaction of trans-permethrin in microsomal incubations was the cleavage of ester linkage to give PBalc, followed by oxidation to 4'-OH-PBalc, 4'-OH-PBacid, and PBacid. As to 4'-hydroxylation of PBalc, several CYPs were able to catalyze the reaction, and CYP2E1 was identified as a predominant isoform. PBacid and its conjugates (glucuronide and glycine) are major urinary metabolites of trans-permethrin in mammals. PBacid is also a metabolite of several pyrethroids, and has been used as a biomarker of human exposure to pyrethroids. Our study indicated that there was no difference in glucuronyltransferase activity of PBacid between humans and rats, and that only UGT1A9 can catalyze the glucuronidation of PBacid among human UGTs. Some UGT1A9 variants are known to have poor glucuronidation activity. From these results, it was assumed that deficiency or polymorphism of UGT1A9 might affect the profile of PBacid and its conjugates in urine collected from persons exposed to trans-permethrin or other pyrethroids. These results are helpful for understanding the metabolism of trans-permethrin in humans and determining methods for quantification of target analytes for assessment of human exposure to trans-permethrin and other pyrethroids that give PBacid and its conjugates as urinary metabolites.

Absorption, tissue distribution, excretion, and metabolism of clothianidin in rats.

Absorption, distribution, excretion, and metabolism of clothianidin [(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] were investigated after a single oral administration of [nitroimino-(14)C]- or [thiazolyl-2-(14)C]clothianidin to male and female rats at a dose of 5 mg/kg of body weight (bw) (low dose) or 250 mg/kg of bw (high dose). The maximum concentration of carbon-14 in blood occurred 2 h after administration of the low oral dose for both labeled clothianidins, and then the concentration of carbon-14 in blood decreased with a half-life of 2.9-4.0 h. The orally administered carbon-14 was rapidly and extensively distributed to all tissues and organs within 2 h after administration, especially to the kidney and liver, but was rapidly and almost completely eliminated from all tissues and organs with no evidence of accumulation. The orally administered carbon-14 was almost completely excreted into urine and feces within 2 days after administration, and approximately 90% of the administered dose was excreted via urine. The major compound in excreta was clothianidin, accounting for >60% of the administered dose. The major metabolic reactions of clothianidin in rats were oxidative demethylation to form N-(2-chlorothiazol-5-ylmethyl)-N'-nitroguanidine and the cleavage of the carbon-nitrogen bond between the thiazolylmethyl moiety and the nitroguanidine moiety. The part of the molecule containing the nitroguanidine moiety was transformed mainly to N-methyl-N'-nitroguanidine, whereas the thiazol moiety was further metabolized to 2-(methylthio)thiazole-5-carboxylic acid. With the exception of the transiently delayed excretion of carbon-14 at the high-dose level, the rates of biokinetics, excretion, distribution, and metabolism of clothianidin were not markedly influenced by dose level and sex.

Mobility and degradation of the herbicide imazosulfuron in lysimeters under field conditions.

The mobility and degradation of imazosulfuron, labeled with carbon-14 at the imidazole ([imi-(14)C]imazo) or pyrimidine ring ([pyr-(14)C]imazo), in lysimeters with 1 m(2) surface and 110 cm depth were investigated for three years. One lysimeter was treated with [imi-(14)C]imazo in two successive years at the rate of 50 g of active ingredient (ai)/ha each. The other two lysimeters were treated once with [pyr-(14)C]imazo and a mixture (1:1, w/w) of the two labeled imazosulfurons, respectively (50 g of ai/ha). In the first and second years of monitoring, the yearly mean concentration of (14)C in the leachate water was <0.10 microg/L in each lysimeter. Although in the third year the concentration of (14)C in the leachate water was 0.17 microg/L for the lysimeter treated twice with [imi-(14)C]imazo, the concentration of imazosulfuron and its degradation products in the leachate water ranged from 0.01 to 0.06 microg/L. At the study termination, the main portion of (14)C recovered was found in the upper 30 cm soil layer in each lysimeter, and no (14)C was detected below a depth of 50 cm. These findings indicated that imazosulfuron and its degradation products in soils translocated into groundwater only slightly.