Effects of coronavirus disease 2019 on the spread of respiratory-transmitted human-to-human bacteria.

OBJECTIVES: The coronavirus disease 2019 (COVID-19) pandemic has necessitated significant changes in medical systems, social behaviours, and non-pharmaceutical interventions (NPIs). We aimed to determine the effect of the COVID-19 pandemic on changes in the epidemiology of respiratory-transmitted bacteria that have been unexplored. METHODS: We utilised a comprehensive national surveillance database from 2018 to 2021 to compare monthly number of patients with four respiratory-transmitted human-to-human bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes) before and after the COVID-19 pandemic, stratified by specimen sources and age groups. RESULTS: The incidence of detected patients with S. pneumoniae, H. influenzae, and S. pyogenes from both respiratory and blood cultures significantly decreased from 2019 to 2020. In 2021, the incidence of detected patients with the respiratory-transmitted bacterial species, except for S. pyogenes, from respiratory cultures, increased again from April to July, primarily affecting the 0-4-year age group. CONCLUSIONS: Our comprehensive national surveillance data analysis demonstrates the dynamic changes and effects of NPIs on respiratory-transmitted bacteria during the COVID-19 pandemic, with variations observed among species, specimen sources, and age groups.

In vitro activity of cefiderocol against carbapenemase-producing and meropenem-non-susceptible Gram-negative bacteria collected in the Japan Antimicrobial Resistant Bacterial Surveillance.

OBJECTIVES: The treatment options available for infections caused by multidrug-resistant Gram-negative pathogens are often limited. Cefiderocol (CFDC) is a novel siderophore cephalosporin that exhibits activity against these pathogens. Several studies have reported the in vitro activity of CFDC against isolates from Europe, the United States, and China, but the activity against carbapenem-resistant bacteria with IMP-type carbapenemase has not been extensively studied. We, therefore, studied the in vitro activities of CFDC against carbapenem-resistant bacteria with available genomic backgrounds based on whole-genome sequencing (WGS) in Japan, where the IMP-type is the predominant carbapenemase produced by Gram-negative rods. METHODS: We selected 603 isolates (528 Enterobacterales, 18 Pseudomonas aeruginosa, and 57 Acinetobacter spp.) from a collection of Gram-negative clinical isolates collected during a Japan Antimicrobial Resistance Bacterial Surveillance program and evaluated the antimicrobial activities of CFDC, ceftolozane/tazobactam (CTLZ/TAZ), imipenem-relebactam (IPM/REL), and ceftazidime/avibactam (CAZ/AVI) against carbapenemase-producing Enterobacterales, carbapenemase-non-producing meropenem-non-susceptible Enterobacterales, and carbapenemase-producing nonfermentative bacteria. RESULTS: Among these, 97.7% of carbapenemase-producing Enterobacterales (99.2% of IMP-type carbapenemase-producing Enterobacterales), 100% of carbapenemase-producing P. aeruginosa, and 91.2% of carbapenemase-producing Acinetobacter spp. were susceptible to CFDC, showing better antimicrobial activity than the other antimicrobial agents evaluated in this study. CFDC was highly effective against class A-, B-, and D ß-lactamase-harbouring isolates when compared to the other antimicrobial agents. In addition, the relationship between CFDC resistance and three genetic factors involved in resistance was discussed. CONCLUSIONS: This is the first large-scale study to systematically demonstrate the efficacy of CFDC against IMP-type carbapenemase-producing strains with known genomic backgrounds.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

Exploring the effects of antimicrobial treatment on the gut and oral microbiomes and resistomes from elderly long-term care facility residents via shotgun DNA sequencing.

Monitoring antibiotic-resistant bacteria (ARB) and understanding the effects of antimicrobial drugs on the human microbiome and resistome are crucial for public health. However, no study has investigated the association between antimicrobial treatment and the microbiome-resistome relationship in long-term care facilities, where residents act as reservoirs of ARB but are not included in the national surveillance for ARB. We conducted shotgun metagenome sequencing of oral and stool samples from long-term care facility residents and explored the effects of antimicrobial treatment on the human microbiome and resistome using two types of comparisons: cross-sectional comparisons based on antimicrobial treatment history in the past 6 months and within-subject comparisons between stool samples before, during and 2-4 weeks after treatment using a single antimicrobial drug. Cross-sectional analysis revealed two characteristics in the group with a history of antimicrobial treatment: the archaeon Methanobrevibacter was the only taxon that significantly increased in abundance, and the total abundance of antimicrobial resistance genes (ARGs) was also significantly higher. Within-subject comparisons showed that taxonomic diversity did not decrease during treatment, suggesting that the effect of the prescription of a single antimicrobial drug in usual clinical treatment on the gut microbiota is likely to be smaller than previously thought, even among very elderly people. Additional analysis of the detection limit of ARGs revealed that they could not be detected when contig coverage was <2.0. This study is the first to report the effects of usual antimicrobial treatments on the microbiome and resistome of long-term care facility residents.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales.

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum ß-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC ß-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Distribution, Trends, and Antimicrobial Susceptibility of Bacteroides, Clostridium, Fusobacterium, and Prevotella Species Causing Bacteremia in Japan During 2011-2020: A Retrospective Observational Study Based on National Surveillance Data.

Background: The increasing prevalence of anaerobic bacteremia is a major concern worldwide and requires longitudinal monitoring. Methods: We present one of the largest and longest longitudinal studies on the prevalence and antimicrobial resistance of Bacteroides, Clostridium, Fusobacterium, and Prevotella spp. isolated from blood culture samples using national comprehensive surveillance data in Japan during 2011-2020 as part of the Japan Nosocomial Infections Surveillance. Results: Data for 41 949 Bacteroides spp., 40 603 Clostridium spp., 7013 Fusobacterium spp., and 5428 Prevotella spp. isolates were obtained. The incidences of bacteremia caused by Bacteroides fragilis, Clostridium perfringens, and Fusobacterium nucleatum significantly increased during the period (P < .0001). Among the 20 species analyzed, 18 showed no significant changes in susceptibility over time, including B. fragilis, C perfringens, and F. nucleatum. However, resistance to clindamycin increased in B. thetaiotaomicron (P = .0312), and resistance to ampicillin increased in B. ovatus (P = .0008). Conclusions: Our comprehensive national surveillance data analysis demonstrated a continuous increase in the incidence of anaerobic bacteremia, particularly in B. fragilis, C. perfringens, and F. nucleatum. This may be linked to the increasing number of colorectal cancer cases or advancing methods for species identification and susceptibility testing, requiring cautious interpretation. The discovery of an upsurge in anaerobic bacteremia and potential alterations in susceptibility highlights the necessity for more extensive studies in this field.

Association between the proportion of laparoscopic approaches for digestive surgeries and the incidence of consequent surgical site infections, 2009-2019: A retrospective observational study based on national surveillance data in Japan.

BACKGROUND: Surgical site infections (SSIs) are among the most common healthcare-associated infections. Laparoscopy is increasingly being used in various surgical procedures. However, no study has examined the association between the proportion of laparoscopic procedures and the incidence of SSIs in digestive surgery using nationwide surveillance data. METHODS: We retrospectively investigated national SSI surveillance data from the Japan Nosocomial Infections Surveillance between 2009 and 2019. The annual trend of the SSI rate and the proportion of laparoscopic procedures were assessed, focusing on five major digestive surgeries. This was based on data from 109,544 (appendix surgery), 206,459 (gallbladder surgery), 60,225 (small bowel surgery), 363,677 (colon surgery), and 134,695 (rectal surgery) procedures. The effect of a 10% increase in the proportion of laparoscopic procedures on the reduction of the SSI rate was estimated using mixed-effect logistic regression. FINDINGS: The average SSI rate of the five digestive surgeries decreased from 11.8% in 2009 to 8.1% in 2019. The proportion of laparoscopic procedures in each of the five digestive surgeries increased continuously (p<0.001). The SSI rate for laparoscopic procedures was always lower than that for open procedures. The results were consistent between all and core hospitals participating in the surveillance. The odds ratios of the 10% increase in the proportion of laparoscopic procedures for five digestive surgeries were always <0.950 (p<0.001). CONCLUSION: An increase in the proportion of laparoscopic procedures was associated with a reduction in the SSI rate in digestive surgeries.

Different CprABC amino acid sequences affect nisin A susceptibility in Clostridioides difficile isolates.

Clinical isolates of Clostridioides difficile sometimes exhibit multidrug resistance and cause diarrhea after antibiotic administration. Metronidazole and vancomycin are often used as therapeutic agents, but resistance to these antibiotics has been found clinically. Therefore, the development of alternative antimicrobial agents is needed. Nisin A, produced by Lactococcus lactis, has been demonstrated to be effective against C. difficile infection. In this study, we evaluated the susceptibility of 11 C. difficile clinical isolates to nisin A and found that they could be divided into 2 groups: high and low susceptibility. Since CprABC and DltDABC, which are responsible for nisin A efflux and cell surface charge, respectively, have been reported to be related to nisin A susceptibility, we investigated the expression of cprA and dltA among the 11 strains. cprA expression in all strains was induced by nisin A, but dltA expression was not. The expression levels of both genes did not correlate with nisin A susceptibility in these clinical isolates. To evaluate cell surface charge, we performed a cytochrome C binding assay and found no relationship between charge and nisin A susceptibility. Then, we determined the whole genome sequence of each clinical isolate and carried out phylogenetic analysis. The 11 isolates separated into two major clusters, which were consistent with the differences in nisin A susceptibility. Furthermore, we found common differences in several amino acids in the sequences of CprA, CprB, and CprC between the two clusters. Therefore, we speculated that the different amino acid sequences of CprABC might be related to nisin A susceptibility. In addition, C. difficile strains could be divided in the same two groups based on susceptibility to epidermin and mutacin III, which are structurally similar to nisin A. These results suggest that genotypic variations in C. difficile strains confer different susceptibilities to bacteriocins.

Disinfectant Susceptibility of Third-Generation-Cephalosporin/Carbapenem-Resistant Gram-Negative Bacteria Isolated from the Oral Cavity of Residents of Long-Term-Care Facilities.

We have recently reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). Since disinfectants are often used in the oral cavity, it is important to investigate the disinfectant susceptibility of oral bacteria. Here, we evaluated the susceptibilities of Gram-negative antimicrobial-resistant bacteria (GN-ARB), including Pseudomonas, Acinetobacter, and Enterobacteriaceae, obtained from the oral cavity of residents of LTCFs to povidone-iodine (PVPI), cetylpyridinium chloride (CPC), benzalkonium chloride (BZK), and chlorhexidine chloride (CHX). We also evaluated the susceptibilities of isolates from the rectum to the same agents to compare the susceptibility profiles of oral and rectal isolates. Next, we investigated the relationship between their susceptibility and disinfectant resistance genes delineated by whole-genome sequencing of the isolates. Additionally, we evaluated the correlation between disinfectant-resistant GN-ARB and clinical information. In oral GN-ARB, the MIC of PVPI showed almost identical values across isolates, while the MICs of CPC, BZK, and CHX showed a wide range of variation among species/strains. In particular, Pseudomonas aeruginosa exhibited high-level resistance to CPC and BZK. The disinfectant susceptibility of rectal GN-ARB showed a tendency similar to that of oral GN-ARB. The presence of qacEΔ1 was correlated with CPC/BZK resistance in P. aeruginosa, while other species exhibited no correlation between qacEΔ1 and resistance. Multiple analyses showed the correlation between the presence of CPC-resistant bacteria in the oral cavity and tube feeding. In conclusion, we found that some oral GN-ARB isolates showed resistance to not only antibiotics but also disinfectants. IMPORTANCE Antibiotic-resistant bacteria (ARB) are becoming a serious concern worldwide. We previously reported the isolation of third-generation-cephalosporin-resistant Gram-negative bacteria from the oral cavity of residents of a long-term-care facility (LTCF). To prevent infection with ARB in hospitals and eldercare facilities, we must pay more attention to the use of not only antibiotics but also disinfectants. However, the effect of disinfectants on ARB is unclear. In this study, we evaluated the susceptibility of Gram-negative ARB (GN-ARB) from the oral cavity of residents of LTCFs to some disinfectants that are often used for the oral cavity; we found that some isolates showed resistance to several disinfectants. This is the first comprehensive analysis of the disinfectant susceptibility of oral GN-ARB. These results provide some important information for infection control and suggest that disinfectants should be applied carefully.

Oral and Rectal Colonization by Antimicrobial-Resistant Gram-Negative Bacteria and Their Association with Death among Residents of Long-Term Care Facilities: A Prospective, Multicenter, Observational, Cohort Study.

INTRODUCTION: The prevalence of antimicrobial-resistant bacteria (ARB) in long-term care facilities (LTCFs) remains unclear. Furthermore, the effect of ARB colonization on the clinical outcomes of LTCF residents has not been explored. METHODS: We conducted a prospective multicenter cohort study and investigated the residents (N = 178) of six Japanese LTCFs (three Welfare Facilities for the Elderly Requiring Long-term Care and three Geriatric Health Service Facilities) for oral and rectal carriage of ARB. The clinical outcomes of the residents were evaluated based on isolating bacterial strains and subjecting them to whole-genome sequencing. RESULTS: Of the 178 participants, 32 belonging to Geriatric Health Service Facilities with no information on their clinical outcome were excluded, and the remaining 146 were followed up for at most 21 months. Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales and Pseudomonas aeruginosa were detected in 42.7% (n = 76) and 2.8% (n = 5) of the rectal swabs and 5.6% (n = 10) and 3.4% (n = 6) of the oral swabs, respectively. Detection of ARB in the oral and rectal cavities showed remarkable association with enteral nutrition. Further, P. aeruginosa was significantly associated with an increase in mortality of the residents, but there were not significant association between ESBL-producing Enterobacterales and mortality. Core-genome phylogeny of P. aeruginosa revealed a wide-spread distribution of the isolated strains across the phylogeny, which included a cluster of ST235 strains with substantially higher biofilm formation ability than the other isolated P. aeruginosa strains. DISCUSSION/CONCLUSION: This study is the first to investigate the carriage of both oral and rectal ARB, genomic relatedness and determinants of antimicrobial resistance in isolated strains, and clinical outcomes of LTCF residents. Our study provides the first direct evidence for the burden of antimicrobial resistance in LTCFs.

Distribution, trends, and antifungal susceptibility of Candida species causing candidemia in Japan, 2010-2019: A retrospective observational study based on national surveillance data.

The increasing incidence of candidemia and the emergence of drug-resistant Candida species are major concerns worldwide. Therefore, long-term surveillance studies are required. Here, we provide one of the largest longitudinal overviews of the trends in the prevalence of Candida species using national data of 57 001 candidemia isolates obtained from > 2000 hospitals for the 2010-2019 period in the Japan Nosocomial Infections Surveillance database. The proportion of Candida species, except Candida krusei and Candida guilliermondii, was almost the same during the study period. The proportion of C. guilliermondii surpassed that of C. krusei in 2014. The incidence of candidemia due to C. albicans (P < 0.0001), C. parapsilosis (P = 0.0002), and C. tropicalis (P < 0.0001) have decreased significantly over this period. Azole susceptibility of C. tropicalis was low, with 17.8% of isolates resistant to fluconazole and 13.5% resistant to voriconazole. The micafungin susceptibility of C. glabrata was low, with 8.0% of isolates showing resistance. The resistance rate of C. krusei toward amphotericin B fluctuated considerably (between 3.2% and 35.7%) over this period. The incidence rate of candidemia caused by C. parapsilosis and C. guilliermondii in hospitals responsible for bone marrow transplantation was significantly higher than that in other hospitals. Overall, our study suggests that in Japan, the species distribution of Candida was almost the same in this period and similar to that reported in North America and Europe. A relatively high resistance to azoles and micafungin was observed in C. glabrata, C. tropicalis, and C. krusei isolates, which require continued surveillance.

This study verifies that the proportion of Candida species in Japan was almost the same from 2010­2019. A relatively higher resistance to azoles and micafungin was observed for C. glabrata, C. tropicalis, and C. krusei isolates.

Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-ß1.

BACKGROUND: Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-ß1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-ß1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated. METHODS: The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-ß1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro. RESULTS: Compared to control lung fibroblasts, pirfenidone significantly restored TGF-ß1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-ß1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1. CONCLUSIONS: Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.

Specific Features of Fibrotic Lung Fibroblasts Highly Sensitive to Fibrotic Processes Mediated via TGF-ß-ERK5 Interaction.

BACKGROUND/AIMS: Lung fibrosis is associated with lung tissue contraction due to abnormal accumulation of myofibroblasts, which aggressively promote the fibrotic process. Transforming growth factor (TGF)-ß signaling in fibroblasts promotes extracellular matrix (ECM) synthesis and fibroblast migration and differentiation into myofibroblasts. Inhibition of extracellular signal-regulated kinase (ERK)5 blocks lung fibroblast activation by suppressing TGF-ß signaling. Here, we examined the effects of an ERK5 inhibitor on TGF-ß1-induced fibrosis in lung fibroblasts. METHODS: The effects of ERK5 inhibition following TGF-ß1 exposure were evaluated in lung fibroblasts isolated from fibrotic human lung tissues. Fibroblast-mediated collagen gel contraction and fibroblast migration towards fibronectin were assessed. Phenotypic differences in fibrotic fibroblasts were examined using the cap analysis gene expression method for genome-wide quantification of promoter activity. RESULTS: TGF-ß1stimulated contraction of collagen gels, fibroblast migration, and α-smooth muscle actin and fibronectin expression, and Smad3 phosphorylation were increased in fibrotic fibroblasts as compared to normal lung fibroblasts. Treatment with the ERK5 inhibitor blocked these responses to a greater extent in fibroblasts from patients with usual interstitial pneumonia as compared to nonspecific interstitial pneumonia, independent of bone morphogenetic protein/Smad1 regulation. Moreover, 223 genes including fibulin-5 -which is involved in the TGF-ß1-ERK5 signaling network- were upregulated in fibrotic fibroblasts, and ECM regulation was found to be enriched in the Reactome analysis. CONCLUSION: ERK5 inhibition attenuated the high sensitivity of fibrotic fibroblasts to TGF-ß1/Smad3 signaling. Thus, the ERK5 pathway components and fibulin-5 are potential therapeutic targets to prevent lung fibrosis progression.

Maser: one-stop platform for NGS big data from analysis to visualization.

Database URL: http://cell-innovation.nig.ac.jp/maser/.

Reproductive isolation between 2 karyotypes in natural populations of the leaf beetle Chrysolina aurichalcea.

Japanese populations of the leaf beetle, Chrysolina aurichalcea, have 2 main karyotypes, Type 31 and Type 41, which greatly differ in chromosome number and form. We examined their geographical distribution and reproductive isolation in southern Hokkaido, northern Japan. Type 41 was widely distributed in the survey area, whereas Type 31 was restricted to a narrow portion. A transect across each karyotypic habitat revealed that both karyotypes co-occurred in a approximately 5 km wide zone, with few hybrids or hybrid derivatives. Assessment of the fine scale distribution showed that the 2 karyotypes do not occupy separate microhabitats within the sympatric zone. Karyotype combinations of copulating pairs in the sympatric zone showed no evidence of assortative mating according to karyotype. These findings suggest that postmating isolation between the 2 karyotypes is strong, whereas premating isolation is weak. It does not agree with results from previous studies, which reported a substantial degree of both post- and premating isolation between the karyotypes. Possible causes of the discrepancy in premating isolation are discussed.

Rapid chromosomal changes inferred from variation in mitochondrial DNA among populations of the leaf beetle Chrysolina aurichalcea (Coleoptera: Chrysomelidae) in Japan.

There are two karyotypes among Japanese populations of the leaf beetle Chrysolina aurichalcea . In "Type 31" beetles, the diploid number of chromosomes is 2n=32 in females and 31 in males, while in "Type 41" individuals, the diploid number of chromosomes is 2n=42 in females and 41 in males. Both karyotypes occur allopatrically or parapatrically in the wild, depending on location; generally, Type 41 beetles inhabit mountainous areas and Type 31 beetles occur on lowland plains. To assess genetic differentiation among C. aurichalcea populations and between the two karyotypes, we collected 606 individuals throughout Japan, determined the karyotypes of 203 individuals, and examined sequence variation in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene for all 606 individuals. Karyotypic analysis indicated that hybrids between the two karyotypes are rare. A haplotype phylogeny shows that neither karyotype is monophyletic. These results may be explained by ancestral polymorphism and/or interkaryotype hybridization. Population genetic analysis showed that genetic divergence between the two karyotypes is relatively low, and similar to variation within karyotypes. Our data suggest that chromosomal changes occurred rapidly and recently. We also discuss the phylogeography of this species and the association between speciation and chromosomal rearrangements.