Genetic characterization of KHM-1 metallo-ß-lactamase-producing Enterobacterales isolates from inpatient sources in Osaka, Japan.

OBJECTIVES: KHM-1-metallo-ß-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii. METHODS: These isolates were characterized by whole-genome sequencing, comparative analysis of blaKHM-1-encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests. RESULTS: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from blaKHM-1 on their chromosomes and plasmids. In all four isolates, blaKHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as blaKHM-1-ISEc68, class I integron cassette, and fosG, the four blaKHM-1-encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions. CONCLUSION: Because no epidemiological contact was observed among the inpatients, the blaKHM-1-encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion.

Comparative whole-genome analysis of diphtheria toxin-producing Corynebacterium ulcerans isolates from sheltered cats in Osaka, Japan.

Diphtheria toxin-producing Corynebacterium ulcerans is a zoonotic pathogen that causes human diphtheria-like symptoms. After performing whole-genome analysis of the five isolates from sheltered cats in Osaka, Japan, we compared them with genome sequences of 25 strains of C. ulcerans from a public database. The five isolates from cats harbored 14 genes encoding possible virulence factors in diphtheria-toxin-producing C. ulcerans. These isolates also had diphtheria toxin gene-encoding prophage in their chromosome, although differences were found in other prophages possession. Whole-genome single-nucleotide polymorphism analysis showed that cats' isolates belonged to ST337 branch, as were strains from Japanese human patients, with 41 or more single-nucleotide polymorphisms variations. High-resolution single-nucleotide polymorphism analysis of C. ulcerans was sufficient to distinguish cats' isolates clearly as not different by conventional genotyping methods.

Hepatitis C Virus-associated Cryoglobulinemic Livedo Reticularis Improved with Direct-acting Antivirals.

We herein report a case of hepatitis C virus (HCV)-associated cryoglobulinemic livedo reticularis in a woman in her 60s that improved with direct-acting antivirals (DAAs). Hyperpigmentation was observed in both lower legs, and a skin biopsy confirmed livedo reticularis, suggesting a relationship with cryoglobulinemia and HCV infection. DAAs with an NS5A inhibitor+NS3/4A protease inhibitor (glecaprevir/pibrentasvir) were administered for eight weeks, and a sustained virological response (SVR) was obtained. The disappearance of serum cryoglobulin was confirmed approximately two years after an SVR was obtained and livedo reticularis was improved. DAA therapy can be an effective therapeutic option for extrahepatic complications associated with HCV infection.

Long-Term Effects of Rifaximin on Patients with Hepatic Encephalopathy: Its Possible Effects on the Improvement in the Blood Ammonia Concentration Levels, Hepatic Spare Ability and Refractory Ascites.

Background and Objectives: To investigate the long-term efficacy of rifaximin (RFX) for hyperammonemia and efficacy for refractory ascites in patients with cirrhosis. Materials and Methods: We enrolled 112 patients with liver cirrhosis who were orally administered RFX in this study. Changes in the clinical data of patients were evaluated up to 36 months after RFX administration. The primary endpoint was a change in blood ammonia levels. Secondary endpoints included changes in clinical symptoms, Child−Pugh (CP) score, number of hospitalizations, degree of refractory ascites, adverse events, and the relationship between RFX administration and the renin-angiotensin-aldosterone system. Results: An improved rate of overt hepatic encephalopathy (HE) of 82.7% was observed 3 months after RFX administration, which significantly induced a progressive decrease in blood ammonia concentration and an improved CP score up to 36 months. No serious RFX treatment-related adverse events were observed. 36.5% in patients after RFX administration improved refractory ascites. After RFX administration, patients with satisfactory control of hepatic ascites without addition of diuretic had lower renin concentration than those with poor control (p < 0.01). At less than 41 pg/mL renin concentration, the control of refractory ascites was significantly satisfactory (p < 0.0001). Conclusions: RFX reduced blood ammonia concentration and improved hepatic spare ability and the quality of life of patients with long-term HE to up to 36 months. Our study revealed the effects of RFX against refractory ascites, suggesting that renin concentration may be a predictive marker for assessing ascites control.

Comparative whole-genome sequence analysis of a BoNT/B5-producing Clostridium botulinum isolate from an infant botulism case of unknown source in Osaka, Japan.

A case of infant botulism of unknown origin, not involved in honey consumption, occurred in Osaka, Japan in 2020. A Clostridium botulinum type B strain named Osaka2020 was isolated from a stool sample of the patient. To clarify the epidemiology of the case, we performed whole-genome sequencing (WGS) of the isolate and compared it with strains from other sources. WGS analysis revealed that isolate Osaka2020 was classified into ST133 of a new sequence type, B5 subtype, and its toxin gene was encoded in a ∼274 kb plasmid. This plasmid was closely related to the pCLJ plasmid from strain 657Ba in the USA, reported to be conjugatively transferable to other strains. Moreover, isolate Osaka2020 also possesses another smaller plasmid that was common with some type A(B) infant botulism isolates in Japan. The phylogenetic tree from whole-genome SNP analysis showed that isolate Osaka2020 was the most closely related to a type B infant botulism isolate that occurred in Japan 10 years ago. Although no epidemiological connection among the two cases was confirmed, there is possibility that the cases are attributed to common causes such as some environmental substance.

Infiltration of hidden antimicrobial resistance among healthy people in a Japanese community.

Background: Under non-antimicrobial selective pressure, antimicrobial-resistant bacteria do not easily become dominant in the microbiota. Furthermore, their low levels prevent detection by isolation, resulting in an underestimation of the prevalence of antimicrobial-resistant bacteria. Objectives: We evaluated the infiltration of antimicrobial-resistant bacteria and their related ß-lactamase genes among healthy people in non-clinical settings. Methods: Cephalosporin- and fluoroquinolone-resistant Escherichia coli and bla genes were quantified in 217 faecal samples from healthy people in non-clinical settings in Japan. E. coli colonies grown on deoxycholate hydrogen sulphide-lactose (DHL) agar, with and without antimicrobials (cefotaxime and ciprofloxacin), were quantified, and E. coli isolates were analysed for their susceptibility to antimicrobials and the presence of bla genes. DNA extracted from faecal samples was used to quantify bla genes using quantitative PCR (qPCR). Results: The isolation rates of cefotaxime- and ciprofloxacin-resistant E. coli were 6.9% and 12.4%, respectively, using agars without antimicrobials, and 12.0% and 24.4%, respectively, using agars with antimicrobials. For samples from which cefotaxime- and ciprofloxacin-resistant E. coli were isolated only using agars with antimicrobials, the ratios of cfu on DHL agars with and without antimicrobials were below -2 log. E. coli harbouring bla genes were isolated from 35.0% of the faecal samples using agars, and bla genes were detected in 65.0% of faecal DNA samples using qPCR. Conclusions: Among people carrying cefotaxime- and ciprofloxacin-resistant E. coli in non-clinical settings, cefotaxime- and ciprofloxacin-resistant E. coli were not dominant in half of the subjects. These individuals may play a role as reservoirs of antimicrobial-resistant bacteria.

Rapid detection and discrimination of potentially toxigenic Corynebacterium ulcerans and Corynebacterium pseudotuberculosis by multiplex real-time PCR and amplicon melting curve analysis.

We developed a multiplex real-time PCR assay with amplicon melting curve analysis to rapidly discriminate Corynebacterium ulcerans from Corynebacterium pseudotuberculosis and detect the bacterial diphtheria toxin gene. This assay should be a valuable tool for identification of potentially toxigenic C. ulcerans.

Eosinophilic Granuloma of the Liver Mimicking Metastatic Liver Tumor.

We herein report a case of coagulation necrosis with granulation and eosinophilic infiltration of the liver. A 37-year-old woman was diagnosed with a new mass lesion in the liver 1 month after breast cancer surgery and admitted for a further examination. Because the tumor occurred immediately after surgery, it was considered essential to determine whether or not it was a metastatic liver tumor from breast cancer. A percutaneous liver tumor biopsy revealed eosinophilic granuloma of the liver, which is considered to have a high possibility of visceral larva migrans with suspected gnathostomiasis infection. A detailed medical history and histological diagnosis are important for making a differential diagnosis.

High production of egc2-related staphylococcal enterotoxins caused a food poisoning outbreak.

Staphylococcal enterotoxins (SEs) produced by enterotoxigenic Staphylococcus aureus in food cause staphylococcal food poisoning. We recently reported a foodborne outbreak due to S. aureus harboring new SE/SE-like (SEl) genes (seg, sei, sem, sen, seo, and selu) related to enterotoxin gene cluster (egc) 2 as with other research groups. However, the pathogenicity of SEs production remains unclear. Therefore, we herein investigated egc2-related SEs production from S. aureus isolates and leftover food items during a foodborne outbreak using a sandwich enzyme-linked immunosorbent assay suitable for the quantification of SEs. S. aureus isolates produced markedly high levels of egc2-related SEs, and the leftover food item "Sushi" contained SEs over the toxin dose that causes food poisoning symptoms. A representative isolate was subjected to whole-genome sequencing. The isolate was homologous with previously reported ST45 strains, particularly the unique genomic island νSaß structure mostly consisting of egc2. The present study indicates that egc2-related SEs are food poisoning causative agents based on high SE production levels within an actual foodborne outbreak.

Novel Endoscopic Therapy for Gastric Varices Using Direct Forward-Viewing Endoscopic Ultrasonography.

Gastric varices (GV) carry a high risk of massive hemorrhage because of potential rupture. To reduce the risk associated with GV, patients need to undergo hemostatic and preventive treatment. The objective of this retrospective study was to evaluate the usefulness of a new method, direct forward-viewing endoscopic ultrasonography (DFV-EUS) for the treatment of GV. We performed endoscopic injection sclerotherapy with histoacryl (EIS-HA) using DFV-EUS for GV in four patients. The paracentesis success rate was 75% (3/4). DFV-EUS has a significant advantage for the treatment of GV in that it can show physicians endoscopic and ultrasound views in real time during the delivery of the sclerosant into the GV. However, the proper use of the ultrasound view must be elucidated through further research for safer and more effective therapy. In the presence of distance between the mucosal surface and vascular lumen or when the blood flow site requires puncture as an additional treatment, DFV-EUS might be a good candidate for the treatment of GV. Altogether, EIS-HA with DFV-EUS might be a new therapeutic option for patients with GV.

Seven-year surveillance of the prevalence of antimicrobial-resistant Escherichia coli isolates, with a focus on ST131 clones, among healthy people in Osaka, Japan.

OBJECTIVES: Escherichia coli (E. coli) is an indicator of antimicrobial resistance, and some strains of E. coli cause infectious diseases. E. coli sequence type 131 (ST131) - a global antimicrobial-resistant pandemic E. coli clone - is frequently detected in clinical specimens. Antimicrobial-resistant bacteria are monitored via national surveillance in clinical settings; however, monitoring information in non-clinical settings is limited. This study elucidated antimicrobial resistance trends of E. coli and dissemination of ST131 among healthy people in non-clinical settings. METHODS: This study collected 517 E. coli isolates from healthy people in Osaka, Japan, between 2013 and 2019. It analysed antimicrobial susceptibility of the isolates and detected the bla and mcr genes in ampicillin-resistant and colistin-resistant isolates, respectively, and the ST131 clone. RESULTS: Antimicrobial resistance rates of the bacteria isolated from healthy people in non-clinical settings were lower than for those in clinical settings. The resistance of the isolates to cefotaxime (4.4%) and ciprofloxacin (13.5%) gradually increased during the study period. In 23 cefotaxime-resistant isolates, the most frequent bla genes belonged to the blaCTX-M-9 group, followed by blaCTX-M-1 goup, blaTEM and blaCMY-2. One mcr-1-harbouring colistin-resistant isolate was detected in 2016. The incidence of the E. coli O25b-ST131 clone was approximately 5% until 2015 and 10% after 2016. CONCLUSION: Both ciprofloxacin resistance and O25b-ST131 clone frequency increased during the study period. Antimicrobial-resistant bacteria gradually spread in healthy people in non-clinical settings; one reason behind this spread was dissemination of global antimicrobial-resistant pandemic clones.

Genomic characterization of clinical Enterobacter roggenkampii co-harbouring blaIMP-1- and blaGES-5-encoding IncP6 and mcr-9-encoding IncHI2 plasmids isolated in Japan.

OBJECTIVES: The spread of carbapenemase-producing Enterobacterales (CPE) with colistin resistance is a critical public health issue. We genetically characterized the clinical isolate Enterobacter roggenkampii OIPH-N260, which harboured carbapenemase genes blaIMP-1 and blaGES-5 with multiple resistance genes, including mcr-9 and blaCTX-M-9. METHODS: This isolate was characterized by whole-genome sequencing, comparative analysis of resistance plasmids, susceptibility tests, bacterial conjugation, S1-nuclease digested pulsed-field-gel electrophoresis, and Southern blot hybridization. RESULTS: The OIPH-N260 isolate exhibited resistance to most ß-lactams and colistin. It co-harboured two resistance plasmids, the blaIMP-1- and blaGES-5-encoding IncP6 plasmid pN260-3 and mcr-9- and blaCTX-M-9-encoding IncHI2 plasmid pN260-1. The comparative analysis of pN260-3 indicated that a unique blaIMP-1-surrounding region was inserted into the blaGES-5-encoding plasmid with the mobile element IS26, which plays an important role in the spread of resistance genes. pN260-1 did not possess the mcr-9 expression regulative gene qseBC. Both plasmids were transferable into other bacterial species via conjugation. CONCLUSIONS: This is the first study to report not only a blaIMP-1 and blaGES-5 co-encoding plasmid, but also the co-harbouring of another plasmid carrying mcr-9 and blaCTX-M-9 in Enterobacter cloacae complex. The development of advanced resistance via IS26-mediated insertion and the co-harbouring of resistance plasmids highlights the need to monitor for resistance genes in CPE.

Irsogladine maleate alters expression of a tight junction protein in portal hypertensive gastropathy.

BACKGROUND AND AIM: Portal hypertensive gastropathy (PHG) is characterized by noninflammatory edema and vasodilatation of the lamina propria of the mucosal epithelium. In addition, the alterations of intercellular junction proteins and dilatation of the endothelial gaps have been reported. In this study, we examined whether irsogladine maleate (IM), a gastric mucosal protective agent, has the potential to improve PHG by restoration of tight junctions (TJs). METHODS: Twenty-four patients with PHG were registered and randomly assigned into two groups: 12 patients in the IM-administration group and 12 patients in the non-administration group. In the administration group, IM (4 mg/day) was administered orally for 12 weeks. Gastric mucosa with a red color in patients with PHG were obtained endoscopically on the registration day and 12 weeks later. The endoscopic findings were evaluated, an immunohistochemical analysis of claudin-3 (a TJ protein) expression in gastric mucosal tissues by a laser microscope was performed, and claudin-3 expression was quantified by western blot analysis. RESULTS: Irsogladine maleate improved the degree of PHG in 2/12 patients endoscopically, in contrast to none of the 12 patients in the non-administration group. Immunohistochemical analysis showed that expression of claudin-3 increased in 8/12 patients in the IM-administration group and 2/12 patients in the non-administration group (P = 0.036). Western blot analysis revealed that the increase in claudin-3 after 12 weeks was significantly higher in the IM-administration group than in the non-administration group (P = 0.010). CONCLUSIONS: The present pilot study suggested that IM might improve the gastric mucosa in PHG through restoration of TJ-protein claudin-3.

Hepatitis B virus core-related antigen is useful for surveillance of hepatocellular carcinoma recurrence in a patient with occult hepatitis B virus infection: Case report.

Serum HBV core-related antigen (HBcrAg) is useful for detecting HCC in patients with occult HBV infection. Surveillance for HCC is needed in patients who are positive for HBcrAg, even if they are negative for HBsAg and HBV DNA.

Prevalence and mechanisms of fluoroquinolone-resistant Escherichia coli among sheltered companion animals.

To better understand the prevalence of fluoroquinolone-resistant Escherichia coli among sheltered companion animals, we conducted a screening study of 38 dogs and 78 cats and investigated the resistance mechanisms and characteristics of the isolates. Fluoroquinolone-resistant E. coli was detected in 18 dogs (47.4 %) and 14 cats (17.9 %). The isolates carried one to four mutations in the gyrA, parC and parE genes of the quinolone resistance-determining region, and the number of mutations was proportional to the MIC for ciprofloxacin. For plasmid-mediated quinolone resistance, aac-(6')-Ib-cr was detected in nine isolates, qnrS in five isolates and qnrB in one isolate. A relationship between the presence of these genes and MIC for ciprofloxacin was not apparent. Statistical analysis indicated that fluoroquinolone-resistant E. coli was widely distributed among sheltered companion animals with various attributes. This may relate to the wide dissemination of fluoroquinolone resistance among humans and other animals in Japan.

Exploratory Research on the Relationship between Human Gut Microbiota and Portal Hypertension.

Objective The relationship between gut microbiota and portal hypertension remains unclear. We investigated the characteristics of the gut microbiota in portal hypertension patients with esophago-gastric varices and liver cirrhosis. Methods Thirty-six patients (12 patients with portal hypertension, 12 healthy controls, and 12 non-cirrhosis patients) were enrolled in this university hospital study. Intestinal bacteria and statistical analyses were performed up to the genus level using the terminal restriction fragment length polymorphism method targeting 16S ribosomal RNA genes, with diversified regions characterizing each bacterium. Results Levels of Lactobacillales were significantly higher (p=0.045) and those of Clostridium cluster IV significantly lower (p=0.014) in patients with portal hypertension than in other patients. This Clostridium cluster contains many butanoic acid-producing strains, including Ruminococcace and Faecalibacterium prausnitzii. Clostridium cluster IX levels were also significantly lower (p=0.045) in portal hypertension patients than in other patients. There are many strains of Clostridium that produce propionic acid, and the effects on the host and the function of these bacterial species in the human intestine remain unknown. Regarding the Bifidobacterium genus, which is supposed to decrease as a result of cirrhosis, no significant decrease was observed in this study. Conclusion In the present study, we provided information on the characteristics of the gut microbiota of portal hypertension patients with esophago-gastric varices due to liver cirrhosis. In the future, we aim to develop probiotic treatments following further analyses that include the species level, such as the intestinal flora analysis method and next-generation sequencers.

Capnocytophaga felis sp. nov. isolated from the feline oral cavity.

Four strains, KC07070T, KC07105, 11 025B-8C and 11 026B-8-C, were isolated from the oral cavity of cats in 2007 or 2011 in Japan. These strains were Gram-stain-negative rods, exhibited gliding motility, grew in air with 5 % CO2 and showed catalase and oxidase activity. The sequences of 16S rRNA genes of the four strains were 100 % identical. Additionally, the sequences of 16S rRNA genes of KC07070T had identity to those of the type strains of Capnocytophaga canimorsus (97.7 %), Capnocytophaga cynodegmi (97.8 %) and Capnocytophaga canis (97.4 %) and 91.2-93.8% identity to those of other species of the genus Capnocytophaga. The major cellular fatty acids of KC07070T were iso-C15 : 0 (56.2 %) and summed feature 11 (14.9 %). The G+C content of the DNA from KC07070T was 35.6 mol%, and the genome size was 2.88 Mbp. KC07070T had digital DNA-DNA hybridization (dDDH) values of 26.2-27.6% and average nucleotide identity (ANI) values of 75.4-83.3 % to the type strains of the closest relatives, C. canimorsus, C. cynodegmi and C. canis. These results of phylogenetic analysis of 16S rRNA gene sequence, cellular fatty acids compositions and dDDH and ANI values indicate that strain KC07070T represents a novel species, for which we propose the name Capnocytophaga felis sp. nov., with type strain KC07070T (=JCM 32681T=DSM 107251T).

Two Cases of Hepatocellular Carcinoma Arising Over 20 Years after a Sustained Virologic Response Following Interferon Therapy for Chronic Hepatitis C.

The development of hepatocellular carcinoma (HCC) after a sustained virologic response (SVR) due to interferon (IFN) therapy for hepatitis C virus infection remains a serious problem. We herein report 2 cases of HCC that developed more than 20 years after SVR with IFN therapy for chronic hepatitis C. The patients were 89- and 72-year-old men with HCC that developed 24-25 years after an SVR with IFN therapy. These patients regularly underwent imaging examinations; therefore, the HCC was detected in the early stage, when it was still curable. Both cases suggest that long-term surveillance after an SVR is effective for the detection of HCC, and radical treatment is possible.

Hepatitis B Virus Reactivation after Receiving Cancer Chemotherapy under Administration of Leuprorelin Acetate.

An 88-year-old man was admitted for elevated liver enzyme levels. Nine years earlier, the patient had been diagnosed with diffuse large B-cell lymphoma (DLBCL) and undergone rituximab, cyclophosphamide, doxorubicin hydrochloride, oncovin, prednisone (R-CHOP) therapy. This patient previously had had a hepatitis B virus (HBV) infection before chemotherapy. After the chemotherapy, he was administered an luteinizing hormone-releasing hormone (LHRH) agonist for prostate cancer. We diagnosed him with HBV reactivation because of positive serum HBV-DNA. HBV reactivation can occur a long time after chemotherapy, particularly if another treatment with immunity-altering drugs is added. In such cases, additional surveillance may be required to detect HBV reactivation.

Molecular characterization of blaKHM-1 encoding plasmid in an Enterobacter hormaechei subsp. hoffmannii isolate from blood culture.

KHM-1 was first reported in 1997 in Japan as a novel metallo-ß-lactamase mediated by Citrobacter freundii carrying pKHM-1 plasmid. There have been few reports in the clinical field since then. A blaKHM-1-positive Enterobacter hormaechei subsp. hoffmannii in E. cloacae complex, isolate OIPH-N069 was isolated from an inpatient blood culture in 2016. The isolate was characterized by whole-genome sequencing, comparative analysis of the blaKHM-1 encoding plasmid, antimicrobial susceptibility tests, and bacterial conjugation. OIPH-N069 was classified into ST78 of E. cloacae complex, and was multidrug resistant because of the presence of antimicrobial resistance genes in addition to blaKHM-1 on its chromosome and plasmids. blaKHM-1 was located on 136,816 bp of the IncA/C2 plasmid pN069-1, which could be transferred to different bacterial species. The backbone structure, genetic arrangement of the class 1 integron cassette, and the blaKHM-1 gene located downstream of the IncA/C2 antibiotic resistance island, ARI-A, in pN069-1 and pKHM-1 were identical. Horizontal gene transfer of the blaCTX-M-2-ISEcp1 resistance gene module only occurred with pN069-1. The study findings indicate not only the structural conservation of blaKHM-1 encoding plasmids over time and across species, but also the risk of the spread of blaKHM-1 encoding plasmids to other bacterial species and the accumulation of additional resistance genes.