Relationship of Metabolic Dysfunction-Associated Steatohepatitis-Related Hepatocellular Carcinoma with Oral and Intestinal Microbiota: A Cross-Sectional Pilot Study.

Background and Objectives: The incidence of metabolic dysfunction-associated steatohepatitis (MASH)-related hepatocellular carcinoma (HCC) is increasing worldwide, alongside the epidemic of obesity and metabolic syndrome. Based on preliminary reports regarding the potential association of HCC and periodontitis, this study aimed to analyze the involvement of periodontal bacteria as well as the oral and intestinal bacterial flora in MASH-related HCC (MASH-HCC). Materials and Methods: Forty-one patients with MASH and nineteen with MASH-HCC participated in the study, completing survey questionnaires, undergoing periodontal examinations, and providing samples of saliva, mouth-rinsed water, feces, and peripheral blood. The oral and fecal microbiome profiles were analyzed by 16S ribosomal RNA sequencing. Bayesian network analysis was used to analyze the causation between various factors, including MASH-HCC, examinations, and bacteria. Results: The genus Fusobacterium had a significantly higher occupancy rate (p = 0.002) in the intestinal microflora of the MASH-HCC group compared to the MASH group. However, Butyricicoccus (p = 0.022) and Roseburia (p < 0.05) had significantly lower occupancy rates. The Bayesian network analysis revealed the absence of periodontal pathogenic bacteria and enteric bacteria affecting HCC. However, HCC directly affected the periodontal bacterial species Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella intermedia in the saliva, as well as the genera Lactobacillus, Roseburia, Fusobacterium, Prevotella, Clostridium, Ruminococcus, Trabulsiella, and SMB53 in the intestine. Furthermore, P. gingivalis in the oral cavity directly affected the genera Lactobacillus and Streptococcus in the intestine. Conclusions: MASH-HCC directly affects periodontal pathogenic and intestinal bacteria, and P. gingivalis may affect the intestinal bacteria associated with gastrointestinal cancer.

Emergence of carbapenem-resistant Pseudomonas alcaligenes and Pseudomonas paralcaligenes clinical isolates with plasmids harbouring bla IMP-1 in Japan.

Introduction. The emergence of carbapenem-resistant Pseudomonas species producing metallo-ß-lactamase (MBL) has become a serious medical problem worldwide. IMP-type MBL was firstly detected in 1991 in Japan. Since then, it has become one of the most prevalent types of MBLs.Hypothesis/Gap statement. Avirulent species of Pseudomonas, such as Pseudomonas alcaligenes, function as reservoirs of drug resistance-associated genes encoding carbapenemases in clinical settings.Methodology. Active surveillance for carbapenem-resistant Gram-negative pathogens was conducted in 2019 at a hospital in Tokyo, Japan. Of the 543 samples screened for carbapenem-resistant isolates, 2 were species of Pseudomonas. One was from a stool sample from a medical staff member, and the other was from a stool sample from a hospitalized patient.Results. Whole-genome sequencing showed that the former isolate was a strain of P. alcaligenes, and the latter was a strain of Pseudomonas paralcaligenes, a species close to P. alcaligenes. Both isolates were resistant to all carbapenems and harboured bla IMP-1 genes encoding IMP-1 MBL, which conferred resistance to carbapenems. The bla IMP-1 genes of P. alcaligenes and P. paralcaligenes were located on the plasmids, pMRCP2, 323125 bp in size, and pMRCP1333, 16592 bp in size, respectively. The sequence of 82 % of pMRCP2 was 92 % similar to the sequence of a plasmid of P. aeruginosa PA83, whereas the sequence of 79 % of pMRCP1333 was >95 % similar to the sequence of a plasmid of Achromobacter xylosoxidans FDAARGOS 162. The genomic environments surrounding the bla IMP-1 of pMRCP2 and pMRCP1333 differed completely from each other.Conclusions. These results indicate that the two isolates acquired bla IMP-1 from different sources and that P. alcaligenes and P. paralcaligenes function as vectors and reservoirs of carbapenem-resistant genes in hospitals.

Pseudomonas paralcaligenes sp. nov., isolated from a hospitalized patient.

A Gram-stain-negative, aerobic, rod-shaped, non-endospore-forming bacterium, designated as strain MRCP1333T, was isolated from a faecal sample from a hospital patient in Japan. MRCP1333T grew at temperatures of 15-40 °C (optimum 25-35 °C), with 1.0-3.0 % (w/v, 171-513 mM) NaCl [optimum 1-2 % (w/v), 171-342 mM], and at pH 6.0-9.5 (optimum pH 7.0-8.0). The results of phylogenetic analysis based on the sequences of the 16S rRNA gene and the 53 genes encoding the bacterial ribosome protein subunits indicated that MRCP1333T represented a member of the Pseudomonas aeruginosa group, most closely related to Pseudomonas alcaligenes. Whole-genome comparisons, using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that MRCP1333T represented a distinct species in the P. aeruginosa group. Phenotypic characterization tests demonstrated utilization by this strain of citrate, glycerol, and d-malic acid, the ability to reduce nitrite to nitrogen and the ability of this strain to grow in the presence of minocycline and tetrazolium blue, distinguishing this strain from P. alcaligenes and other closely related species of the P. aeruginosa group. The major fatty acids of MRCP1333T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 38.4 %), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.1 %) and C16 : 0 (20.6 %). The DNA G+C content of MRCP1333T was 66.5 mol%. Genetic and phenotypic evidence indicated that MRCP1333T should be classified as representing a novel species, for which the name Pseudomonas paralcaligenes sp. nov. is proposed. The type strain is MRCP1333T (=LMG 32254T,=JCM 34250T).

Increased co-expression of stromal HHLA2 and fibroblast activation protein in upper tract urothelial carcinoma.

BACKGROUND: The human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2; also known as B7 homolog 7 [B7-H7]) regulates immune responses. However, its immunoregulatory role in upper tract urothelial carcinoma (UTUC) remains unclear. METHODS: We evaluated the immunohistochemical expression of HHLA2 and fibroblast activation protein (FAP), which is a marker of cancer-associated fibroblasts, in UTUC tissues from 85 patients who underwent nephroureterectomy. The associations between the expressions of HHLA2 and FAP and clinicopathological characteristics were investigated. RESULTS: The increased expression of HHLA2 in tumor cells (t-HHLA2) was associated with a low histological grade, a negative lymphovascular invasion (LVI), and a low neutrophil-to-lymphocyte ratio, whereas an increased expression of HHLA2 in stromal cells (s-HHLA2) was associated with a high histological grade. No correlation was observed between the expression of t-HHLA2 and s-HHLA2. FAP was expressed only in the stromal cells (s-FAP). Positive s-FAP expression was significantly associated with increased s-HHLA2 expression, higher histological grade, higher pathological T stage, and positive LVI. Higher t-HHLA2 was associated with longer cancer-specific and progression-free survival. In contrast, positive s-FAP was associated with short progression-free survival. CONCLUSION: These findings suggest that the progression of UTUC may involve increased co-expression of HHLA2 and FAP in the tumor stroma.

Circulating tumor cells and cell-free tumor DNA analyses in urothelial cancer using the LiquidBiopsy platform.

Background: Emerging data suggested that liquid biopsy such as detection of circulating tumor cells (CTCs) and cell-free tumor DNA analysis augments the management of patients with urothelial cancer (UC). We presented our pilot experience of liquid biopsy using the Ion Torrent platform to detect CTCs and genomic alterations in UC. Materials and methods: Blood or urine samples from 16 patients were subjected to CTC and plasma/urine cell-free tumor DNA isolation for next generation sequencing (NGS) using the Ion S5 system to detect mutations among 50 oncogenes on the Ion AmpliSeq Cancer Hotspot Panel. Results: The Ion Torrent platform detected a higher number of CTCs than those in previous studies using the CellSearchTM system. Overall, mutations were detected in 13/16 (81.3%) patients with a median number of 18 (range 12-25). NGS isolated 17 hotspot mutations from 11 genes and 41 novel genomic alterations from 24 genes, some of which are supposed to be clinically actionable. Conclusions: The Ion Torrent platform efficiently detected CTCs compared with previous reports. NGS with the present system also allowed for detection of gene alterations which are likely to be therapeutic targets and provided an attractive tool to guide personalized therapy for patients with advanced UC.

Redox Homeostasis is Disturbed by Redox Cycling between Reactive Cysteines of Thioredoxin 1 and 9,10-Phenanthrenequinone, an Atmospheric Electron Acceptor.

9,10-Phenanthrenequinone (9,10-PQ) is a toxicant in diesel exhaust particles and airborne particulate matter ≤2.5 µm in diameter. It is an efficient electron acceptor that readily reacts with dithiol compounds in vitro, resulting in the oxidation of thiol groups and concomitant generation of reactive oxygen species (ROS). However, it remains to be elucidated whether 9,10-PQ interacts with proximal protein dithiols. In the present study, we used thioredoxin 1 (Trx1) as a model of proteins with reactive proximal cysteines and examined whether it reacts with 9,10-PQ in cells and tissues, thereby affecting its catalytic activity and thiol status. Intratracheal injection of 9,10-PQ into mice resulted in protein oxidation and diminished Trx activity in the lungs. Using recombinant wild-type and C32S/C35S Trx1, we found that Cys32 and Cys35 selectively serve as electron donor sites for redox reactions with 9,10-PQ that lead to substantial inhibition of Trx activity. Addition of dithiothreitol restored the Trx activity inhibited by 9,10-PQ. Exposure of cultured cells to 9,10-PQ caused intracellular reactive oxygen species generation that led to protein oxidation, Trx1 dimerization, p38 phosphorylation, and apoptotic cell death. Overexpression of Trx1 blocked these 9,10-PQ-mediated events. These results suggest that the interaction of the reactive cysteines of Trx1 with 9,10-PQ causes oxidative stress, leading to disruption of redox homeostasis.

Association of Nrf2 expression and mutation with Weiss and Helsinki scores in adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignant tumor. Genetic abnormalities that may represent therapeutic targets and prognostic factors in ACC remain unclear. Besides being one of the main cellular defense mechanisms that regulates antioxidant pathways for detoxifying reactive oxygen species (ROS), the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) promotes tumor proliferation by increasing metabolic activity. In surgical specimens from 12 cases of nonmetastatic ACCs and nine cases of benign adrenocortical adenoma (ACA), we investigated gene mutation and protein expressions for Nrf2 and the preoperative maximum standard glucose uptake (SUVmax) on [18 F]fluorodeoxy-glucose positron emission tomography. Three of five ACCs with a Weiss score of 7 to 9 were Nrf2 mutants; these ACCs had higher expression of Nrf2 and higher preoperative SUVmax. The other seven ACCs had a Weiss score of 3 to 6; these seven ACCs and all the ACAs were non-Nrf2 gene mutants. Patients with a Weiss score of 7 to 9 and Nrf2 mutant ACC had shorter overall survival. Based on Helsinki scoring, three ACCs with a Helsinki score greater than 17 had Nrf2 mutants, higher expression of Nrf2, higher preoperative SUVmax, and shorter overall survival. Our findings indicate that Nrf2 activation and the associated increase in metabolism play roles in ACC, in particular in ACC with a Weiss score of 7 to 9 and a Helsinki score of greater than 17.

Increased expression of Nrf2 and elevated glucose uptake in pheochromocytoma and paraganglioma with SDHB gene mutation.

BACKGROUND: Pheochromocytomas (PCC) and paragangliomas (PGL) are catecholamine-producing neuroendocrine tumors. According to the World Health Organization Classification 2017, all PCC/PGL are considered to have malignant potential. There is growing evidence that PCC/PGL represent a metabolic disease that leads to aerobic glycolysis. Cellular energy metabolism involves both transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and succinate dehydrogenase (SDH) subtypes, but the association of these substances with PCC/PGL is largely unknown. METHODS: We investigated SDHB gene mutation and protein expressions for SDHB and Nrf2 in surgical specimens from 29 PCC/PGL. We also assessed preoperative maximum standard glucose uptake (SUVmax) on [18F]fluorodeoxy-glucose positron emission tomography and mRNA levels for Nrf2. RESULTS: Among 5 PCC/PGL with a PASS Score ≥ 4 or with a moderately to poorly differentiated type in the GAPP Score, 4 were metastatic and found to be SDHB mutants with homogeneous deletion of SDHB protein. SDHB mutants showed a higher expression of Nrf2 protein and a higher preoperative SUVmax than non-SDHB mutants with a PASS < 4 or a well-differentiated GAPP type. Furthermore, protein expression of Nrf2 was positively associated with preoperative SUVmax. The Nrf2 mRNA level positively correlated with malignant phenotype, higher expression for Nrf2 protein and SDHB gene mutant, but negatively correlated with expression for SDHB protein. There was also a positive correlation between Nrf2 mRNA level and SUVmax. CONCLUSION: These results suggest that activation of Nrf2 and elevated metabolism play roles in PCC/PGL with malignant potential that have SDHB gene mutation and SDHB deficiency.

Elevated expression of B7 homolog 4 is associated with disease progression in upper urinary tract urothelial carcinoma.

BACKGROUND: B7 homolog 4 (B7-H4) is a negative regulator of immune responses, but its immunoregulatory role in the tumor microenvironment of upper urinary tract urothelial carcinoma (UTUC) remains unclear. METHODS: We measured the immunohistochemical expression of B7-H4, CD8 and T cell intracellular antigen 1 (TIA-1), a marker of activated CD8, in 133 patients with UTUC who underwent nephroureterectomy. We also studied the relationship between B7-H4, CD8 and TIA-1 expression and clinicopathological characteristics. RESULTS: B7-H4 was mainly expressed on the surface in tumor cells, while CD8 and TIA-1 were often expressed in tumor-infiltrating lymphocytes. Elevated expression of B7-H4 in tumor cells was associated with a poorer histological grade, higher pT stage, regional lymph node metastasis, lymphovascular invasion, poorer response of recurrent metastatic lesions to systemic chemotherapy and shorter overall survival. Expression of CD-8 or TIA-1 alone did not correlate directly with clinicopathological characteristics, but among the patients with higher B7-H4 expression in the primary tumors, those with higher CD8 or TIA-1 expression had a better response to systemic chemotherapy, and longer survival, than these with lower CD8 or TIA-1 expression. Cox multivariate regression analysis revealed that higher expression of B7-H4 was associated with shorter overall survival. CONCLUSIONS: These findings suggest that B7-H4 expression in the tumor microenvironment influences the progression of UTUC through cancer immunity and metabolic activity. Tumor cell-associated B7-H4 might be a potential target for cancer immunotherapies.

A transferrable IncL/M plasmid harboring a gene encoding IMP-1 metallo-ß-lactamase in clinical isolates of Enterobacteriaceae.

BACKGROUND: The worldwide spread of carbapenemase-producing Enterobacteriaceae (CPE) has reduced the clinical utility of carbapenems. Plasmids often play an important role in the spread of genes encoding drug-resistance factors, especially in the horizontal transfer of these genes among species of Enterobacteriaceae. This study describes a patient infected with three species of CPE carrying an identical transferrable IncL/M plasmid. METHODS: Clinical isolates of CPE were collected at St. Luke's International Hospital, Tokyo, Japan, from 2015 to 2019. Three species of CPE isolates, Enterobacter cloacae, Klebsiella aerogenes and Serratia marcescens, were isolated from a patient who developed severe gallstone pancreatitis associated with bloodstream infection, with all three isolates producing IMP-1 metallo-ß-lactamase. The complete sequences of the plasmids of the three isolates were determined by both MiSeq and MinION. The medical chart of this patient was retrospectively reviewed conducted to obtain relevant clinical information. RESULTS: The three CPE species carried an IncL/M plasmid, pSL264, which was 81,133 bp in size and harbored blaIMP-1. The genetic environment surrounding blaIMP-1 consisted of int1-blaIMP-1-aac(6')-IIc-qacL-qacEdelta1-sul1-istB-IS21. Conjugation experiments showed that S. marcescens could transmit the plasmid to E. cloacae and K. aerogenes. In contrast, pSL264 could not transfer from E. cloacae or K. aerogenes to S. marcescens. CONCLUSION: The IncL/M plasmid pSL264 harboring blaIMP-1 was able to transfer among different species of Enterobacteriaceae in a patient receiving long-term antimicrobial treatment. The worldwide emergence and spread of IncL/M plasmids harboring carbapenemase-encoding genes among species of Enterobacteriaceae is becoming a serious public health hazard.

Single nucleotide variants of succinate dehydrogenase A gene in renal cell carcinoma.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is mainly associated with a mutation in the SDHB gene and sometimes with mutations in the SDHC or SDHD genes. However, only three cases of succinate dehydrogenase A (SDHA)-deficient RCC have been reported, and the relation between SDHA mutations and RCC has not been clarified. This study assessed the role of SDHA gene mutations in human RCC. We investigated SDHA/B/C/D gene mutations in 129 human RCCs. Targeted next-generation sequencing and direct Sanger sequencing revealed single nucleotide variants (SNVs) of the SDHA gene with amino acid sequence variations in 11/129 tumors, while no SDHB/C/D gene mutations were found. Tumor cells with SNVs of the SDHA gene were characterized by eosinophilic cytoplasm and various patterns of proliferation. Immunohistochemistry examination found that the 11 tumors with SNVs of the SDHA gene showed significant reduction of SDHA protein and SDHB protein expression compared to the 19 tumors without SDHA or SDHB mutations (both P < .0001). Western blotting showed a greater decrease in the expression of SDHA and SDHB proteins in the 11 tumors with SNVs of the SDHA gene than in the 19 tumors without (both P < .0001). There was a positive correlation between SDHA and SDHB protein levels (P < .0001). On immunohistochemistry and Western blotting, the 11 tumors with SNVs of the SDHA gene had higher protein expression for nuclear factor E2-related factor 2 (Nrf2) compared to the 19 tumors without the mutation (P < .01). These observations suggest that SDHA gene mutations might be associated with a subset of RCC.

Bacteroidetes Species Are Correlated with Disease Activity in Ulcerative Colitis.

Fecal microbiota transplantation following triple-antibiotic therapy (amoxicillin/fosfomycin/metronidazole) improves dysbiosis caused by reduced Bacteroidetes diversity in patients with ulcerative colitis (UC). We investigated the correlation between Bacteroidetes species abundance and UC activity. Fecal samples from 34 healthy controls and 52 patients with active UC (Lichtiger's clinical activity index ≥5 or Mayo endoscopic subscore ≥1) were subjected to next-generation sequencing with HSP60 as a target in bacterial metagenome analysis. A multiplex gene expression assay using colonoscopy-harvested mucosal tissues determined the involvement of Bacteroidetes species in the mucosal immune response. In patients with UC, six Bacteroides species exhibited significantly lower relative abundance, and twelve Bacteroidetes species were found significantly correlated with at least one metric of disease activity. The abundance of five Bacteroidetes species (Alistipes putredinis, Bacteroides stercoris, Bacteroides uniformis, Bacteroides rodentium, and Parabacteroides merdae) was correlated with three metrics, and their cumulative relative abundance was strongly correlated with the sum of Mayo endoscopic subscore (R = -0.71, p = 2 × 10-9). Five genes (TARP, C10ORF54, ITGAE, TNFSF9, and LCN2) associated with UC pathogenesis were expressed by the 12 key species. The loss of key species may exacerbate UC activity, serving as potential biomarkers.

A mucosal immune response induced by oral administration of heat-killed Mycobacterium avium subsp. paratuberculosis exacerbates EAE.

Findings in humans and animals have demonstrated a potential role for Mycobacterium avium subsp. paratuberculosis (MAP) antigenic components in encephalitogenic T cell activation. Here we reported that oral administration of MAP activates the mucosal immunity and exacerbates active experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice, modulating the immune cell traffic from secondary lymphoid organs to central nervous system. The detection of antigenic mycobacterial components by intestinal antigen-presenting cells may modulate the immune system and the subsequent inflammatory status through various signaling mechanisms, including the synthesis of pro-inflammatory cytokines involved in EAE pathogenesis.

Increased expression of adenosine 2A receptors in metastatic renal cell carcinoma is associated with poorer response to anti-vascular endothelial growth factor agents and anti-PD-1/Anti-CTLA4 antibodies and shorter survival.

BACKGROUND: Adenosine and its adenosine 2A receptors (A2AR) mediate the immunosuppressive mechanism by which tumors escape immunosurveillance and impede anti-tumor immunity within the tumor microenvironment. However, we do not know whether the adenosine pathway (CD39/CD73/A2AR) plays a role in renal cell carcinoma (RCC). Therefore, we studied the role of immunosuppression in RCC by assessing the adenosine pathway in patients with RCC treated with anti-vascular endothelial growth factor (anti-VEGF) agents or immune checkpoints inhibitors (ICIs) or both. METHODS: In 60 patients with metastatic RCC, we examined the expression of CD39, CD73, A2AR, and programmed cell death 1 ligand 1 (PD-L1) immunohistochemically in surgically resected tumor tissues and studied the clinicopathological characteristics of these patients. Patients were treated by cytoreductive nephrectomy with systemic therapy with anti-VEGF agent or a combination of the ICIs anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody and programmed cell death 1 (PD-1) antibody. RESULTS: Increased expression of A2AR in the primary tumors was associated with metastatic profiles. Patients treated with anti-PD-1 antibody in monotherapy, a combination of anti-PD-1 and anti-CTLA4 antibodies, or anti-VEGF agents showed better response and longer overall survival if the primary tumor had higher PD-L1 expression and lower A2AR expression. In Cox multivariate regression analysis, higher expression of A2AR was associated with shorter overall survival. CONCLUSIONS: Our findings suggest that the expression of A2AR and PD-L1 in the primary tumors in RCC might predict the outcomes of treatment with anti-VEGF agents and ICIs and that the A2AR pathway might be a molecular target for immunotherapy.

Emergence of Carbapenem-Resistant Providencia rettgeri and Providencia stuartii Producing IMP-Type Metallo-ß-Lactamase in Japan.

Four Providencia rettgeri isolates and one Providencia stuartii isolate were obtained from urine samples of five patients in 2018 in Japan. All of the isolates were resistant to imipenem and meropenem, and three were highly resistant to both carbapenems, with MICs of 512 µg/ml. The three highly carbapenem-resistant isolates harbored blaIMP-70, encoding a variant of IMP-1 metallo-ß-lactamase with two amino acid substitutions (Val67Phe and Phe87Val), and the other two harbored blaIMP-1 and blaIMP-11, respectively. Whole-genome sequencing revealed that an isolate harbored two copies of blaIMP-1 on the chromosome and that the other four harbored a copy of blaIMP-11 or blaIMP-70 in a plasmid. Expression of blaIMP-70 conferred carbapenem resistance in Escherichia coli Recombinant IMP-70 and an IMP-1 variant with Val67Phe but without Phe87Val had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that an amino acid substitution of Val67Phe affects increased activities against meropenem in IMP-70. These results suggest that Providencia spp. become more highly resistant to carbapenems by acquisition of two copies of blaIMP-1 or by mutation of blaIMP genes with amino acid substitutions, such as blaIMP-70.

Pseudomonas yangonensis sp. nov., isolated from wound samples of patients in a hospital in Myanmar.

Strains of a Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated MY50T, MY63 and MY101, were isolated from wound samples of three hospitalized patients in Yangon, Myanmar. Strains MY50T, MY63 and MY101 grew at temperatures of 4-44 °C, in media containing 1.0-7.0 % (w/v) NaCl and at pH 6.0-9.5. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences showed that these strains belonged to the genus Pseudomonas and were part of the Pseudomonas oleovorans group and located close to Pseudomonas guguanensis and Pseudomonas mendocina. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization analyses, confirmed that strains MY50T, MY63 and MY101 were the same strain and they were a distinct species in the P. oleovorans group. Results of phenotypic characterization tests demonstrated that utilization of p-hydroxy-phenylacetic acid, glycerol, l-pyroglutamic acid and quinic acid could distinguish these strains from other species of the P. oleovorans group. These genetic and phenotypic characteristics suggest that they should be classified as representing a novel species, under the proposed name Pseudomonas yangonensis sp. nov. The type strain is MY50T (=LMG 31602T,=JCM 33396T), with a DNA G+C content of 62.82 mol%.

Emergence of carbapenem-resistant and colistin-susceptible Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 in Japan.

BACKGROUND: The spread of Enterobacteriaceae producing both carbapenemases and Mcr, encoded by plasmid-mediated colistin resistance genes, has become a serious public health problem worldwide. This study describes three clinical isolates of Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 that were resistant to carbapenem but susceptible to colistin. METHODS: Thirty-two clinical isolates of E. cloacae complex non-susceptible to carbapenems were obtained from patients at 14 hospitals in Japan. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods and E-tests. Their entire genomes were sequenced by MiSeq and MinION methods. Multilocus sequence types were determined and a phylogenetic tree constructed by single nucleotide polymorphism (SNP) alignment of whole genome sequencing data. RESULTS: All 32 isolates showed MICs of ≥2 µg/ml for imipenem and/or meropenem. Whole-genome analysis revealed that all these isolates harbored blaIMP-1, with three also harboring mcr-9. These three isolates showed low MICs of 0.125 µg/ml for colistin. In two of these isolates, blaIMP-1 and mcr-9 were present on two separate plasmids, of sizes 62 kb and 280/290 kb, respectively. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr-9. In the third isolate, however, both blaIMP-1 and mcr-9 were present on the chromosome. CONCLUSION: The mcr-9 is silently distributed among carbapenem-resistant E. cloacae complex isolates, of which are emerging in hospitals in Japan. To our knowledge, this is the first report of isolates of E. cloacae complex harboring both blaIMP-1 and mcr-9 in Japan.

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats.

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

Emergence and Spread of Carbapenem-Resistant and Aminoglycoside-Panresistant Enterobacter cloacae Complex Isolates Coproducing NDM-Type Metallo-ß-Lactamase and 16S rRNA Methylase in Myanmar.

Surveillance of 10 hospitals and a regional public health laboratory in Myanmar identified 31 isolates of carbapenem-resistant Enterobacter cloacae complex harboring blaNDM-type Of these isolates, 19 were highly resistant to aminoglycosides and harbored one or more genes encoding 16S rRNA methylases, including armA, rmtB, rmtC, and/or rmtE Of the 19 isolates, 16 were Enterobacter xiangfangensis ST200, with armA on the chromosome and a plasmid harboring blaNDM-1 and rmtC, indicating that these isolates were clonally disseminated nationwide in Myanmar.IMPORTANCE The emergence of multidrug-resistant E. cloacae complex has become a public health threat worldwide. E. xiangfangensis is a recently classified species belonging to E. cloacae complex. Here, we report a clonal dissemination of multidrug-resistant E. xiangfangensis ST200 producing two types of New Delhi metallo-ß-lactamase (NDM-type MBL), NDM-1 and -4, and three types of 16S rRNA methylases, ArmA, RmtC, and RmtE, in hospitals in Myanmar. The observation of these multidrug-resistant E. xiangfangensis ST200 isolates stresses the urgency to continue molecular epidemiological surveillance of these pathogens in Myanmar and in South Asian countries.

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates from hospitals in Myanmar.

OBJECTIVE: The aim of this study was to clarify the genetic and epidemiological properties of multidrug-resistant Acinetobacter baumannii in medical settings in Myanmar. METHODS: A total of 45 A. baumannii clinical isolates were obtained in medical settings in Myanmar. The whole genomes were sequenced by a next generation sequencer, and the phylogenetic tree was constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. RESULTS: Thirty-eight MDR Acinetobacter baumannii isolates were obtained from seven hospitals in Myanmar. The majority of MDR A. baumannii isolates belonged to ST2. Of the 38 isolates, 5 harbored blaNDM-1, and 28 did armA or armA2 CONCLUSIONS: A. baumannii ST2 producing 16S rRNA methylase ArmA has been spreading in medical settings in Myanmar.