The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro.

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C.2. Compared with that seen at 37 °C, the intrinsic clearance rates (Vmax/Km) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A, .1B, .10, and .23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and .16 were decreased (79 ∼ 84%), those of CYP3A4.2 and .7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2, .7, .16, and .18 were decreased (58 ∼ 82%).3. These findings may be clinically useful for dose optimisation in patients with hypothermia or hyperthermia.

Changes in Pharmacodynamic Parameters during Co-administration of 5-FU with Warfarin: A Retrospective Case Series.

Drug-drug interactions (DDIs) between warfarin (WF) and fluoropyrimidines are well known. Co-administration of WF and 5-fluorouracil (5-FU) leads to elevations in prothrombin time international normalised ratio (PT-INR). The inhibition of drug metabolism through suppression of CYP activity is a possible cause of prolonged PT-INR elevations. 5-FU and its metabolites are suspected to inhibit CYPs, but the precise mechanisms of action remain unknown. This study aimed to investigate the possible DDI effects of the co-administration of 5-FU with WF using PT-INR and PT-INR/dose ratio as pharmacodynamic parameters. Retrospective case series data were collected from patients who received parenteral 5-FU chemotherapy from April 2009 to December 2019 at the University of the Ryukyus Hospital. Seven patients who received 5-FU in combination with WF were analysed. There was a significant increase in PT-INR and PT-INR/dose during the co-administration of WF and 5-FU (p = 0.0018 and p = 0.0187, respectively; paired t-test). The findings demonstrated significant DDI between 5-FU and WF evident as elevated PT-INR and PT-INR/dose ratio.

Time-dependent inhibition of CYP3A4-mediated midazolam metabolism by macrolide antibiotics in CYP3A4 genetic variants: Comparison with testosterone metabolism.

OBJECTIVES: The present study aimed to evaluate the effects of CYP3A4 genetic variation on the kinetics of mechanism-based inhibition (MBI) of both inhibitors using midazolam as a substrate for comparison with our previous study, as midazolam and testosterone have different binding sites. BACKGROUND: The genetic variation of cytochrome P450 (CYP) 3A4 affects MBI, expressed as the maximum inactivation rate constant (kinact,max) and the inhibitor concentration required to achieve half-maximal inactivation (KI). We previously showed, using testosterone as a substrate, that the MBI kinetics of erythromycin and clarithromycin differ among CYP3A4 variants. MATERIALS AND METHODS: Midazolam 1'-hydroxylation inactivation profiles of erythromycin and clarithromycin were assessed using recombinant CYP3A4.1, .2, .7, .16, and .18 expressed in Escherichia coli. MBI parameters were calculated from changes in the inactivation rate constant (Δkobs) by the inhibitors. RESULTS: Both inhibitors increased Δkobs value in a concentration- and preincubation time-dependent manner, and MBI kinetics differed among variants. Trends of differences in MBI parameters among variants were similar to those assessed using testosterone as a substrate; KI decreased for CYP3A4.7, and kinact,max decreased for CYP3A4.2, .7, and .16. CONCLUSION: The genetic variation of recombinant CYP3A4 affects the MBI profile of CYP3A4 by erythromycin and clarithromycin, while the influence of genetic variation was similarly observed regardless of substrates. Our findings are of clinical relevance because the residual enzyme activity of CYP3A4 in the presence of inhibitor was estimated to vary among genetic variants.

Revised hospital antibiotic formulary reduces antimicrobial consumption and promotes a shift towards narrow-spectrum antibiotic usage.

BACKGROUND: In Japan, the national action plan to adress antimicrobial resistance problems aimed to reduce the use of oral cephalosporins, quinolones, and macrolides per day per 1000 inhabitants by 50% from the levelin 2013 by 2020. The aim of this study was to evaluate the effects of a revised antibiotic formulary on in- and out-hospital oral antibiotic prescribing practices at a 600-bed university hospital. METHOD: A retrospective before-and-after comparison study was conducted. All antimicrobial consumption data in the reviewed classes from 1 January 2013 to 31 December 2018, were extracted from the hospital database's electronic medical records. The data were measured in the defined daily dose and antibiotic use density (defined daily dose per 1000 patient-days). RESULTS: The total oral antibiotic use densities for in-hospital prescriptions in 2013 and 2018 were 117.95 and 75.42, respectively, and 239.83 and 193.88, respectively, for out-hospital prescriptions. From 2013 to 2018, antibiotic use densities of second- and third-generation cephalosporins, macrolides and fluoroquinolones for in-hospital prescriptions changed annually by -49.00%, -92.67%, +0.49% and -48.19%, and out-hospital prescriptions of these antibiotics changed by +76.69%, -86.37%, -16.29% and -51.75%, over the same period. Penicillin prescriptions increased by 71.31% for in-hospital and 42.72% for out-hospital prescriptions over this period. CONCLUSIONS: The revised hospital antibiotic formulary reduced total antibiotic consumption and increased the use of narrow-spectrum antibiotics for both in- and out-hospital prescriptions.

Effects of 6-paradol, an unsaturated ketone from gingers, on cytochrome P450-mediated drug metabolism.

Paradols are unsaturated ketones produced by biotransformation of shogaols in gingers. Among them, 6-paradol has been investigated as a new drug candidate due to its anti-inflammatory, apoptotic, and neuroprotective activities. In this study, the inhibitory effects of 6-paradol on the activities of cytochrome P450 (CYP) enzymes were investigated with human liver microsomes and recombinant CYP isozymes. 6-Paradol showed concentration-dependent inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 isozymes, with IC50 values ranging from 3.8 to 21.4µM in recombinant CYP isozymes. However, the inhibition was not potentiated following pre-incubation, indicating that 6-paradol is not a mechanism-based inhibitor. These results suggest that pharmacokinetic drug-drug interactions might occur with 6-paradol, which must be considered in the process of new drug development.

Inhibitory Effects of Garcinia cambogia Extract on CYP2B6 Enzyme Activity.

This study assessed the inhibitory effects of Garcinia cambogia extract on the cytochrome P450 enzymes in vitro. G. cambogia extract was incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes and recombinant CYP2B6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on cytochrome P450 enzyme activities. The results showed that G. cambogia extract has significant inhibitory effects on CYP2B6 activity in a concentration-dependent manner. Furthermore, the inhibition was potentiated following preincubation with NADPH, indicating that G. cambogia extract is a time-dependent inhibitor of CYP2B6. Meanwhile, hydroxycitric acid, the major bioactive ingredient of G. cambogia extract, did not exhibit significant inhibition effects on cytochrome P450 enzyme activities. G. cambogia extract could modulate the pharmacokinetics of CYP2B6 substrate drugs and lead to interactions with those drugs. Therefore, caution may be required with respect to concomitant intake of dietary supplements containing G. cambogia extract with CYP2B6 substrates.

Generation of enterocyte-like cells with pharmacokinetic functions from human induced pluripotent stem cells using small-molecule compounds.

The small intestine plays an important role in all aspects of pharmacokinetics, but there is no system for the comprehensive evaluation of small-intestinal pharmacokinetics, including drug metabolism and absorption. In this study, we aimed to construct an intestinal pharmacokinetics evaluation system and to generate pharmacokinetically functional enterocytes from human induced pluripotent stem cells. Using activin A and fibroblast growth factor 2, we differentiated these stem cells into intestinal stem cell-like cells, and the resulting cells were differentiated into enterocytes in a medium containing epidermal growth factor and small-molecule compounds. The differentiated cells expressed intestinal marker genes and drug transporters. The expression of sucrase-isomaltase, an intestine-specific marker, was markedly increased by small-molecule compounds. The cells exhibited activities of drug-metabolizing enzymes expressed in enterocytes, including CYP1A1/2, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, UGT, and sulfotransferase. Fluorescence-labeled dipeptide uptake into the cells was observed and was inhibited by ibuprofen, an inhibitor of the intestinal oligopeptide transporter solute carrier 15A1/PEPT1. CYP3A4 mRNA expression level was increased by these compounds and induced by the addition of 1α,25-dihydroxyvitamin D3. CYP3A4/5 activity was also induced by 1α,25-dihydroxyvitamin D3 in cells differentiated in the presence of the compounds. All these results show that we have generated enterocyte-like cells that have pharmacokinetic functions, and we have identified small-molecule compounds that are effective for promoting intestinal differentiation and the gain of pharmacokinetic functions. Our enterocyte-like cells would be useful material for developing a novel evaluation system to predict human intestinal pharmacokinetics.

Establishment and directed differentiation of induced pluripotent stem cells from glycogen storage disease type Ib patient.

Glycogen storage disease type Ib (GSDIb) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), which leads to neutrophil dysfunction. However, the underlying causes of these dysfunctions and their relationship with glucose homeostasis are unclear. Induced pluripotent stem cells (iPSCs) hold a great promise for advances in developmental biology, cell-based therapy and modeling of human disease. Here, we examined the use of iPSCs as a model for GSDIb. In this study, one 2-year-old patient was genetically screened and diagnosed with GSDIb. We established iPSCs and differentiated these cells into hepatocytes and neutrophils, which comprise the main pathological components of GSDIb. Cells that differentiated into hepatocytes exhibited characteristic albumin secretion and indocyanine green uptake. Moreover, iPSC-derived cells generated from patients with GSDIb metabolic abnormalities recapitulated key pathological features of the diseases affecting the patients from whom they were derived, such as glycogen, lactate, pyruvate and lipid accumulation. Cells that were differentiated into neutrophils also showed the GSDIb pathology. In addition to the expression of neutrophil markers, we showed increased superoxide anion production, increased annexin V binding and activation of caspase-3 and caspase-9, consistent with the GSDIb patient's neutrophils. These results indicate valuable tools for the analysis of this pathology and the development of future treatments.

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6ß-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Influence of cytochrome P450 2C19 gene variations on pharmacokinetic parameters of thalidomide in Japanese patients.

PURPOSE: Cytochrome P450 (CYP)2C19 polymorphisms may partly explain the variability of thalidomide concentration and adverse drug effects by altering its metabolism. To compare the genetic and clinical factors responsible for the adverse effects and efficacy of thalidomide treatment, we investigated CYP2C19 genetic polymorphisms in Japanese subjects. MATERIALS AND METHODS: Variations in the CYP2C19 gene in 6 patients treated with thalidomide were analyzed. The dosage of thalidomide, concentrations of (R)- and (S)-thalidomide in whole blood, and clinical laboratory test results were used as pharmacokinetic and pharmacodynamic indices. Using genomic DNA, CYP2C19*2 and *3 allele frequencies were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. RESULTS: The frequencies of CYP2C19 PM and hetero EM (hetEM) genotypes in Japanese patients taking thalidomide were 2 (33.3%) and 4 (66.7%), respectively. The areas under the curve (AUC) of (R)-thalidomide were 3.42 and 5.33 µg·h/L, and those of (S)-thalidomide were 1.64 and 2.46 µg·h/L for hetEM and PM, respectively. CONCLUSIONS: This study provided new insights regarding the contribution of CYP2C19 gene variations to adverse responses to thalidomide. Genotyping of CYP2C19*2 and *3 can be considerably simplified by using KOD FX as a polymerase for prediction of adverse effects to thalidomide by the PCR-RFLP method. CYP2C19 PM patients tend to have high serum thalidomide concentrations.

Virulence of Bursaphelenchus xylophilus Isolated from Naturally Infested Pine Forests to Five Resistant Families of Pinus thunbergii.

Pine wilt disease is one of the most serious epidemic tree diseases in Japan, and resistant pine trees have been developed through a breeding program. To evaluate resistance of resistant families of Japanese black pine, Pinus thunbergii, to the pinewood nematode, Bursaphelenchus xylophilus, isolated from the field, and to determine whether differentiation of pathogenicity to resistant pine families appears in the nematode isolates, seedlings of five resistant pine families were inoculated with 25 nematode isolates. Disease incidence 18 weeks after inoculation was significantly different among nematode isolates and among pine families but there was no interaction effect between nematode isolate and pine family. This indicates that nematode isolates did not have differential host specificity to resistant families of P. thunbergii. Isolate Shimabara, a test isolate of the breeding program, showed the same degree of virulence as the highly virulent isolates frequently used in experiments. However, more virulent isolates than Shimabara were found among the isolates collected from natural pine forest. This indicated that B. xylophilus populations with higher virulence than Shimabara exist in the natural population. These findings are important in development of more efficient breeding procedures for resistant pine trees.

Cytoplasmic incompatibility in the semivoltine longicorn beetle Acalolepta fraudatrix (Coleoptera: Cerambycidae) double infected with Wolbachia.

Wolbachia are obligatory endosymbiotic α-proteobacteria found in many arthropods. They are maternally inherited, and can induce reproductive alterations in the hosts. Despite considerable recent progress in studies on the associations between Wolbachia and various taxonomic groups of insects, none of the researches have revealed the effects of Wolbachia on longicorn beetles as the host insect. Acalolepta fraudatrix is a forest longicorn beetle that is distributed in East Asia. In this study, the relationship between Wolbachia and A. fraudatrix was investigated. Out of two populations of A. fraudatrix screened for Wolbachia using the genes ftsZ, wsp, and 16S rRNA, only one of the populations showed detection of all three genes indicating the presence of Wolbachia. Electron microscopy and fluorescent in situ hybridization also confirmed that the A. fraudatrix population was infected with Wolbachia. Sequencing the wsp genes derived from single insects revealed that two strains of Wolbachia coexisted in the insects based on the detection of two different sequences of the wsp gene. We designated these strains as wFra1 and wFra2. The bacterial titers of wFra1 were nearly 2-fold and 3-fold higher than wFra2 in the testes and ovaries, respectively. The two strains of Wolbachia in the insects were completely eliminated by rearing the insects on artificial diets containing 1% concentration of tetracycline for 1 generation. Reciprocal crosses between Wolbachia-infected and Wolbachia-uninfected A. fraudatrix demonstrated that only eggs produced by the crosses between Wolbachia-infected males and Wolbachia-uninfected females did not hatch, indicating that Wolbachia infecting A. fraudatrix causes cytoplasmic incompatibility in the host insect. This is the first report showing the effect of Wolbachia on reproductive function in a longicorn beetle, A. fraudatrix.

Comparison of the inhibitory profiles of itraconazole and cimetidine in cytochrome P450 3A4 genetic variants.

CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the K(i) value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the K(i) value for CMD was 0.64-fold lower. In CYP3A4.16, the K(i) value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the K(i) value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the K(i) values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates.

Disappearance of summer influenza in the Okinawa prefecture during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic.

Since the Okinawan islands are located in the southernmost part of Japan, where the climate is subtropical, several episodes of influenza epidemics occur during the summer season. More recently, we have demonstrated that summer influenza epidemics occur every year. After the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in January 2020, measures to avoid disease transmission have been widely promoted in Japan, such as the use of masks, handwashing, remote work, and cancellation of large events. These measures might also have reduced the spread of other infectious diseases, such as the seasonal influenza. Based on this background, we evaluated weekly influenza activity in the 2019/2020 season. After the SARS-CoV-2 pandemic, the summer influenza in the Okinawa prefecture disappeared in 2020. The reasons for the disappearance of summer influenza in Okinawa are discussed herein.

Effect of 5-fluorouracil on mRNA expression of drug metabolizing enzyme and transporter genes in human hepatoma cell lines.

Fluoropyrimidines such as 5-fluorouracil (5-FU) are well known to have drug-drug interactions with anticoagulant medications such as warfarin. This study investigated the mRNA expression of pharmacokinetic (PK)-related genes in response to 5-FU using the hepatocarcinoma cell lines after examining relevant gene expression via RNA sequencing. We used HepaRG cells for 5-FU treatment analysis because these cells displayed PK-related gene expression. 5-FU exposure significantly reduced cytochrome P450 3A4 (CYP3A4) mRNA expression. Additionally, the mRNA expression of nuclear receptor subfamily 1 group I member 2 (also known as pregnane X receptor), a nuclear receptor transcription factor that promotes the expression of many CYP genes, was also decreased in HepaRG cells following 5-FU treatment. The mRNA expressions of the CYP2B6 and ATP-binding cassette transporter genes were decreased after 5-FU treatment. This study revealed that 5-FU treatment reduced PK-related gene expression in HepaRG cells. These findings should be useful for further drug-drug interaction research.

Virulence Biomarkers of Bursaphelenchus xylophilus: A Proteomic Approach.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, one of the most serious forest pests worldwide, is considered the causal agent of the pine wilt disease (PWD). The main host species belong to the genus Pinus, and a variation in the susceptibility of several pine species to PWN infection is well-known. It is also recognized that there is variation in the virulence among B. xylophilus isolates. In the present study, we applied a quantitative mass spectrometry-based proteomics approach to perform a deep characterization of proteomic changes across two B. xylophilus isolates with different virulence from different hosts and geographical origins. A total of 1,456 proteins were quantified and compared in the two isolates secretomes, and a total of 2,741 proteins were quantified and compared in the nematode proteomes in pine tree extract and fungus stimuli conditions. From the proteomic analyses, a group of proteins was selected and identified as potential virulence biomarkers and shed light on putative most pathogenic proteins of this plant-parasitic nematode. Proteomic data are available via ProteomeXchange with identifier PXD029377.

Comparison of the inhibitory effects of azole antifungals on cytochrome P450 3A4 genetic variants.

Cytochrome P450 (CYP) 3A4 is one of the major drug-metabolizing enzymes. Genetic variants of CYP3A4 with altered activity are one of the factors responsible for interindividual differences in drug metabolism. Azole antifungals inhibit CYP3A4 to cause clinically significant drug-drug interactions. In the present quantitative study, we investigated the inhibitory effects of three azole antifungals (ketoconazole, voriconazole, and fluconazole) on testosterone metabolism by recombinant CYP3A4 genetic variants (CYP3A4.1 (WT), CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18) and compared them with those previously reported for itraconazole. The inhibition constants (Ki) of ketoconazole, voriconazole, and fluconazole for rCYP3A4.1 were 3.6 nM, 3.2 µM, and 16.1 µM, respectively. The Ki values of these azoles for rCYP3A4.16 were 13.9-, 13.6-, and 6.2-fold higher than those for rCYP3A4.1, respectively, whereas the Ki value of itraconazole for rCYP3A4.16 was 0.54-fold of that for rCYP3A4.1. The other genetic variants had similar effects on the Ki values of the three azoles, whereas a very different pattern was seen for itraconazole. In conclusion, itraconazole has unique characteristics that are distinct from those shared by the other azole anti-fungal drugs ketoconazole, voriconazole, and fluconazole with regard to the influence of genetic variations on the inhibition of CYP3A4.

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae).

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that "Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad-leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus." We used a simple nematode-loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer-using Monochamus beetles after transfer by A. fraudatrix from broad-leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.