High attenuation mucus in bronchi with allergic bronchopulmonary mycosis.

BACKGROUND: High-attenuation mucus (HAM) is a specific manifestation of allergic bronchopulmonary mycosis (ABPM) on chest computed tomography (CT). OBJECTIVES: To compare the diagnostic accuracy of the two definitions of HAM and to clarify the clinical and radiographic characteristics of HAM-positive and HAM-negative ABPM. METHODS: CT images at the diagnosis of ABPM using Asano's criteria were retrospectively analysed. In Study #1, radiographic data obtained using the same CT apparatus in a single institute were analysed to determine the agreement between the two definitions of HAM: a mucus plug that is visually denser than the paraspinal muscles or that with a radiodensity ≥70 Hounsfield units. In Study #2, HAM was diagnosed by comparison with the paraspinal muscles in patients with ABPM reporting to 14 medical institutes in Japan. RESULTS: In Study #1, 93 mucus plugs from 26 patients were analysed. A substantial agreement for HAM diagnosis was observed between the two methods, with a κ coefficient of 0.72. In Study #2, 60 cases of ABPM were analysed; mucus plugs were present in all cases and HAM was diagnosed in 45 (75%) cases. The median A. fumigatus-specific IgE titre was significantly lower in HAM-positive patients than in HAM-negative patients (2.5 vs. 24.3 UA /mL, p = .004). Nodular shadows were observed more frequently in the airways distal to HAM than in those distal to non-HAM mucus plugs (59% vs. 32%, p < .001). CONCLUSION: In conclusion, agreement between the two methods to diagnose HAM was substantial. HAM was associated with some immunological and radiographic characteristics, including lower levels of sensitization to A. fumigatus and the presence of distal airway lesions.

Predictive Value of Various Atypical Cells for the Detection of Human Papillomavirus in Cervical Smears.

It is thought that numerous genotypes of human papillomavirus (HPV) are associated with various atypical cells, such as multinucleated cells, koilocytes, binucleated cells, parakeratotic cells, and giant cells, in the cervix. We previously showed the specificity of HPV genotypes for koilocytes and multinucleated cells. Therefore, in this study, we analyzed the association among HPV genotypes and binucleated cells, parakeratotic cells, and giant cells in Papanicolaou (Pap) smears. We detected HPV genotypes and atypical cells in 651 cases of liquid-based cytology with an abnormal Pap smear. The HPV genotypes associated with atypical cells were evaluated using stepwise logistic regression with backward elimination and a likelihood ratio test for model construction. Polymerase chain reaction was used to determine the HPV genotypes in whole liquid-based cytology samples and microdissected cell samples from Pap smear slides. Binucleated cells were significantly associated with HPV genotype 42. Moreover, parakeratotic cells were significantly associated with certain HPV genotypes, such as HPV40. However, it was difficult to detect specific HPV genotypes by the manual microdissection-polymerase chain reaction method despite the presence of binucleated cells and parakeratotic cells. Thus, the presence of binucleated cells, parakeratotic cells, and giant cells in Pap smears may not be predictive of cervical lesions above low-grade squamous intraepithelial lesions or infection with highly carcinogenic HPV genotypes.

Nine-Year Trend in the Prevalence of Allergic Diseases and Their Associated Factors in Young Adults.

INTRODUCTION: Notably, few studies have evaluated the recent changes in the prevalence of allergic diseases in young adults. Studies examining the risk of allergy in two populations with similar social backgrounds, other than the region in which they live, are rare. METHODS: First-year students from Hokkaido University were enrolled in this study between 2011 and 2019. A questionnaire survey was conducted to determine the annual prevalence of current wheeze, seasonal allergic rhinitis (SAR), and perennial allergic rhinitis (PAR) in nonsmoking young adults. Trends in the presence of these disease conditions were evaluated based on their hometowns (Hokkaido and outside Hokkaido separately) due to the low prevalence of cedar pollen allergies in Hokkaido. The association between these disease conditions and body mass index (BMI) was also assessed. RESULTS: The prevalence of current wheeze and PAR food allergies did not change in both regions. SAR showed a significantly increasing trend; however, the prevalence of SAR was higher among those whose place of origin was not Hokkaido. Current wheeze was positively associated with obesity (p < 0.05), whereas the high prevalence of SAR was not associated with body weight. In contrast, a lean body type was significantly associated with a higher prevalence of PAR (p < 0.05). DISCUSSION/CONCLUSION: The prevalence of current wheeze was stable and that of PAR has decreased over the past 9 years. However, the prevalence of SAR in Hokkaido has been increasing in Japanese young adults. A differential association between current wheeze and BMI was observed when comparing PAR and SAR.

Development of a contacting transwell co-culture system for the in vitro propagation of primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a malignant neoplasm of the central nervous system that is refractory to treatment and has extremely poor prognosis. One factor hindering the development of therapeutic options for PCNSL is its molecular heterogeneity and the extreme difficulty in establishing in vitro cell lines that permit intensive research on this disease. In the present study, we developed a method to propagate PCNSL cells in vitro using a contacting transwell cell culture system involving brain vascular pericytes. The co-culture system was found to recapitulate the tumor microenvironment that is influenced by the biological activity of adjacent pericytes, and to sustain the survival and proliferation of PCNSL cells in vitro. We further delineated the underlying molecular mechanisms and found that the HGF-c-Met axis may be involved in the long-term in vitro culture of PCNSL cells. Moreover, the peptidylprolyl isomerase Pin1 was found to play a key role in PCNSL cell survival and it sustained proliferation through interactions with key transcription factors related to B-cell lymphomagenesis. These results suggest that our in vitro co-culture system is well suited to analyzing the biological and molecular characteristics of PCNSL, and may contribute to the discovery of new therapeutic interventions.

Cytological features of human papillomavirus-infected immature squamous metaplastic cells from cervical intraepithelial neoplasia grade 2.

In reflex cytology, the presence of prominent nucleoli in immature metaplastic squamous cells (IM) may be underdiagnosed due to variations in interpretation. The aim of this study is to identify human papillomaviruses (HPVs) that infect IM clusters in cervical intraepithelial neoplasia 2 (CIN2) on Papanicolaou (Pap) smears to determine the cytological features of lesion-derived cells. Thirty-two patients with a simultaneous diagnosis of CIN2 on biopsy and high-grade squamous intraepithelial lesions (HSIL) on cytology as well as with IM clusters on HSIL smears were included. CIN2 tissues and HSIL and IM clusters on Pap smears were isolated by manual microdissection, and HPV types were identified by PCR-based genotyping. The nuclear area within the IM clusters was also measured. The median nuclear area of HPV-negative IM clusters was 48 µm2 , with a coefficient of variation (CV) of 0.20; those of HPV-positive clusters were 66 µm2 and 0.34, respectively. The cut-off values of the nuclear area and CV for HPV positivity were 62 µm2 and 0.25, respectively. IM clusters composed of cells with a nuclear area of more than twice that of neutrophils or cells with a wide variation in nuclear sizes are likely to be neoplastic cells caused by HPV.

Optimal Papanicolaou Smear Conditions for Manual Microdissection of Single Target Cells.

This study aimed to investigate the optimal conditions for Papanicolaou (Pap) smear to increase the success rate of target cell isolation through manual microdissection (MMD) and prevent cell spread. Pap smears were prepared using an HPV42-positive SurePath™ liquid-based cytology case, and 46 and 50 koilocytes were used in wet and dried Pap smears, respectively, to verify the success rate of target cell isolation using MMD based on the HPV detection rate. During MMD, the microscopic examination of both specimens revealed that cells in dried smears could be easily identified; however, cell debris remained in the surrounding area after MMD. Although it was difficult to observe cells in wet smears, there was no cell debris. When the needle tip was immersed in DNA lysate after cell isolation through MMD, a difference in cell solubility was found between dry and wet smears. HPV42 was detected in 94.7% and 97.4% of dried and wet Pap smears, respectively, via polymerase chain reaction genotyping using lysed cell solution; the detection rates were not significantly different. The isolation of target cells from wet Pap smears using MMD reduced the risk of contamination and increased the success rate of HPV detection. This study might facilitate the identification of new CIN-derived HPV-infected cells using MMD with wet Pap smears.

Applicable predictive factors extracted from peak flow trajectory for the prediction of asthma exacerbation.

BACKGROUND: Real-time asthma exacerbation prediction and acute asthma attack detection are essential for patients with severe asthma. Peak expiratory flow (PEF) exhibits a potential for use in long-term asthma self-monitoring. However, the method for processing PEF calculations remains to be clarified. OBJECTIVE: To develop clinically applicable novel exacerbation predictors calculated using PEF records. METHODS: Previously proposed exacerbation predictors, including the slope of PEF, percentage predicted PEF, percentage best PEF, the highest PEF over the lowest PEF within specific periods, and PEF coefficient of variation, in addition to a novel indicator delta PEF moving average (ΔMA), defined as the difference between 14-day and 3-day average PEF values, along with moving average (MA) adjusted for PEF reference (%ΔMA), were verified using the Hokkaido-based Investigative Cohort Analysis for Refractory Asthma data of 127 patients with severe asthma from whom 73,503 PEF observations were obtained. Receiver operating characteristic curves for all predictors were drawn, and the corresponding areas under the curve (AUCs) were computed. Regression analysis for MA and percentage MA were conducted. RESULTS: The most outstanding performance was shown by ΔMA and %ΔMA, with AUC values of 0.659 and 0.665 in the univariate model, respectively. When multivariate models were incorporated with random intercepts for individual participants, the AUC for ΔMA and %ΔMA increased to 0.907 and 0.919, respectively. CONCLUSION: The MA and percentage MA are valuable indicators that should be considered when deriving predictors from the PEF trajectory for monitoring exacerbations in patients with severe asthma. TRIAL REGISTRATION: The Hokkaido-based Investigative Cohort Analysis for Refractory Asthma was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN ID: 000003254). https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000003917.

Infant case of severe immune thrombocytopenia caused by COVID-19 infection.

Immune thrombocytopenia (ITP) is a common childhood acute autoimmune bleeding disorder caused by numerous viruses and characterized by isolated thrombocytopenia. Although cases of ITP caused by coronavirus disease 2019 (COVID-19) infection have been reported in adults, pediatric reports are limited. We present the case of a 1-year-old girl who developed COVID-19-infection-related ITP with a very low platelet count (0.0 × 104/µL). We searched for COVID-19-related pediatric ITP cases and found 10 other cases, with the majority having platelet counts of <1.0 × 104/µL. Although pediatric ITP cases caused by COVID-19 infection may be severe, further studies are needed.

Promotion of ABCG2 gene expression by neolignans from Piper longum L.

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression.

Pathogen Profiles in Outpatients with Non-COVID-19 during the 7th Prevalent Period of COVID-19 in Gunma, Japan.

The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1-7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.

Molecular Evolutionary Analyses of the Spike Protein Gene and Spike Protein in the SARS-CoV-2 Omicron Subvariants.

To better understand the evolution of the SARS-CoV-2 Omicron subvariants, we performed molecular evolutionary analyses of the spike (S) protein gene/S protein using advanced bioinformatics technologies. First, time-scaled phylogenetic analysis estimated that a common ancestor of the Wuhan, Alpha, Beta, Delta variants, and Omicron variants/subvariants diverged in May 2020. After that, a common ancestor of the Omicron variant generated various Omicron subvariants over one year. Furthermore, a chimeric virus between the BM.1.1.1 and BJ.1 subvariants, known as XBB, diverged in July 2021, leading to the emergence of the prevalent subvariants XBB.1.5 and XBB.1.16. Next, similarity plot (SimPlot) data estimated that the recombination point (breakpoint) corresponded to nucleotide position 1373. As a result, XBB.1.5 subvariants had the 5' nucleotide side from the breakpoint as a strain with a BJ.1 sequence and the 3' nucleotide side as a strain with a BM.1.1.1 sequence. Genome network data showed that Omicron subvariants were genetically linked with the common ancestors of the Wuhan and Delta variants, resulting in many amino acid mutations. Selective pressure analysis estimated that the prevalent subvariants, XBB.1.5 and XBB.1.16, had specific amino acid mutations, such as V445P, G446S, N460K, and F486P, located in the RBD when compared with the BA.4 and BA.5 subvariants. Moreover, some representative immunogenicity-associated amino acid mutations, including L452R, F486V, R493Q, and V490S, were also found in these subvariants. These substitutions were involved in the conformational epitopes, implying that these mutations affect immunogenicity and vaccine evasion. Furthermore, these mutations were identified as positive selection sites. These results suggest that the S gene/S protein Omicron subvariants rapidly evolved, and mutations observed in the conformational epitopes may reduce the effectiveness of the current vaccine, including bivalent vaccines such as mRNA vaccines containing the BA.4/BA.5 subvariants.

Allergic bronchopulmonary aspergillosis with atopic, nonatopic, and sans asthma-Factor analysis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) develops in the presence or absence of asthma, either atopic or nonatopic. We have tried to explore the essential components in the pathogenesis of the disease, which are either consistent and variable according to the presence and type of asthma. METHODS: Non-cystic fibrosis ABPA cases satisfying Asano's criteria were extracted from a prospective registry of ABPA and related diseases in Japan between 2013 and 2023. According to the type of preceding asthma, ABPA was classified into three groups: ABPA sans asthma (no preceding asthma), ABPA with atopic asthma, and ABPA with nonatopic asthma. Exploratory and confirmatory factor analyses were performed to identify the components that determined the clinical characteristics of ABPA. RESULTS: Among 106 cases of ABPA, 25 patients (24%) had ABPA sans asthma, whereas 57 (54%) and 24 (23%) had ABPA with atopic and nonatopic asthma, respectively. Factor analysis identified three components: allergic, eosinophilic, and fungal. Patients with atopic asthma showed the highest scores for the allergic component (p < .001), defined by total and allergen-specific IgE titers and lung opacities, and the lowest scores for the fungal component defined by the presence of specific precipitin/IgG or positive culture for A. fumigatus. Eosinophilic components, including peripheral blood eosinophil counts and presence of mucus plugs/high attenuation mucus in the bronchi, were consistent among the three groups. CONCLUSION: The eosinophilic component of ABPA is considered as the cardinal feature of ABPA regardless of the presence of preceding asthma or atopic predisposition.

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase (RdRp) Region and VP1 Gene in Human Norovirus Genotypes GII.P6-GII.6 and GII.P7-GII.6.

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

Molecular evolutionary analyses of the fusion protein gene in human respirovirus 1.

Few evolutionary studies of the human respiratory virus (HRV) have been conducted, but most of them have focused on HRV3. In this study, the full-length fusion (F) genes in HRV1 strains collected from various countries were subjected to time-scaled phylogenetic, genome population size, and selective pressure analyses. Antigenicity analysis was performed on the F protein. The time-scaled phylogenetic tree using the Bayesian Markov Chain Monte Carlo method estimated that the common ancestor of the HRV1 F gene diverged in 1957 and eventually formed three lineages. Phylodynamic analyses showed that the genome population size of the F gene has doubled over approximately 80 years. Phylogenetic distances between the strains were short (< 0.02). No positive selection sites were detected for the F protein, whereas many negative selection sites were identified. Almost all conformational epitopes of the F protein, except one in each monomer, did not correspond to the neutralising antibody (NT-Ab) binding sites. These results suggest that the HRV1 F gene has constantly evolved over many years, infecting humans, while the gene may be relatively conserved. Mismatches between computationally predicted epitopes and NT-Ab binding sites may be partially responsible for HRV1 reinfection and other viruses such as HRV3 and respiratory syncytial virus.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

CKAP4 is a potential exosomal biomarker and therapeutic target for lung cancer.

Background: Globally, lung cancer causes the most cancer death. While molecular therapy progress, including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), has provided remarkable therapeutic effects, some patients remain resistant to these therapies and therefore new target development is required. Cytoskeleton-associated membrane protein 4 (CKAP4) is a receptor of the secretory protein Dickkopf-1 (DKK1) and the binding of DKK1 to CKAP4 promotes tumor growth via Ak strain transforming (AKT) activation. We investigated if CKAP4 functions as a diagnostic biomarker and molecular therapeutic target for lung cancer. Methods: CKAP4 secretion with exosomes from lung cancer cells and the effect of CKAP4 palmitoylation on its trafficking to the exosomes were examined. Serum CKAP4 levels were measured in mouse xenograft models, and 92 lung cancer patients and age- and sex-matched healthy controls (HCs). The lung cancer tissues were immunohistochemically stained for DKK1 and CKAP4, and their correlation with prognosis and serum CKAP4 levels were investigated. Roles of CKAP4 in the lung cancer cell proliferation were examined, and the effects of the combination of an anti-CKAP4 antibody and osimertinib, a third generation TKI, on anti-tumor activity were tested using in vitro and in vivo experiments. Results: CKAP4 was released from lung cancer cells with exosomes, and its trafficking to exosomes was regulated by palmitoylation. CKAP4 was detected in sera from mice inoculated with lung cancer cells overexpressing CKAP4. In 92 lung cancer patients, positive DKK1 and CKAP4 expression patients showed worse prognoses. Serum CKAP4 positivity was higher in lung cancer patients than in HCs. After surgical operation, serum CKAP4 levels were decreased. CKAP4 overexpression in lung cancer cells promoted in vitro cell proliferation and in vivo subcutaneous tumor growth, which were inhibited by an anti-CKAP4 antibody. Moreover, treatment with this antibody or osimertinib, a third generation TKI, inhibited AKT activity, sphere formation, and xenograft tumor growth in lung cancer cells harboring EGFR mutations and expressing both DKK1 and CKAP4, while their combination showed stronger inhibition. Conclusions: CKAP4 may represent a novel biomarker and molecular target for lung cancer, and combination therapy with an anti-CKAP4 antibody and osimertinib could provide a new lung cancer therapeutic strategy.

Molecular Evolutionary Analyses of the Pseudomonas-Derived Cephalosporinase Gene.

Despite the increasing evidence of the clinical impact of Pseudomonas-derived cephalosporinase (PDC) sequence polymorphisms, the molecular evolution of its encoding gene, blaPDC, remains elusive. To elucidate this, we performed a comprehensive evolutionary analysis of blaPDC. A Bayesian Markov Chain Monte Carlo phylogenetic tree revealed that a common ancestor of blaPDC diverged approximately 4660 years ago, leading to the formation of eight clonal variants (clusters A-H). The phylogenetic distances within clusters A to G were short, whereas those within cluster H were relatively long. Two positive selection sites and many negative selection sites were estimated. Two PDC active sites overlapped with negative selection sites. In docking simulation models based on samples selected from clusters A and H, piperacillin was bound to the serine and the threonine residues of the PDC active sites, with the same binding mode for both models. These results suggest that, in P. aeruginosa, blaPDC is highly conserved, and PDC exhibits similar antibiotic resistance functionality regardless of its genotype.