Search

Objectives: Despite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-DⓇ Kit, β-Lactamase, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL and AmpC genes circulating in the United States. Methods: A diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases genes, was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antimicrobial Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. Results: Results were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. Conclusion: The ARM-DⓇ Kit, β-Lactamase detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.

Taste receptor type 1 (T1r) proteins are responsible for recognizing nutrient chemicals in foods. In humans, T1r2/T1r3 and T1r1/T1r3 heterodimers serve as the sweet and umami receptors that recognize sugars or amino acids and nucleotides, respectively. T1rs are conserved among vertebrates, and T1r2a/T1r3 from medaka fish is currently the only member for which the structure of the ligand-binding domain (LBD) has been solved. T1r2a/T1r3 is an amino acid receptor that recognizes various l-amino acids in its LBD as observed with other T1rs exhibiting broad substrate specificities. Nevertheless, the range of chemicals that are recognized by T1r2a/T1r3LBD has not been extensively explored. In the present study, the binding of various chemicals to medaka T1r2a/T1r3LBD was analyzed. A binding assay for amino acid derivatives verified the specificity of this protein to l-α-amino acids and the importance of α-amino and carboxy groups for receptor recognition. The results further indicated the significance of the α-hydrogen for recognition as replacing it with a methyl group resulted in a substantially decreased affinity. The binding ability to the protein was not limited to proteinogenic amino acids, but also to non-proteinogenic amino acids, such as metabolic intermediates. Besides l-α-amino acids, no other chemicals showed significant binding to the protein. These results indicate that all of the common structural groups of α-amino acids and their geometry in the l-configuration are recognized by the protein, whereas a wide variety of α-substituents can be accommodated in the ligand binding sites of the LBDs.

Here the authors demonstrate that de novo DNA methylation mediated by DNMT3B is regulated by nitric oxide (NO). They also isolate a unique modulator (DBIC) that inhibits S-nitrosylation of DNMT3B, which mitigates cell proliferation and tumorigenic conversion in vivo.

Salt taste sensation is multifaceted: NaCl at low or high concentrations is preferably or aversively perceived through distinct pathways. Cl− is thought to participate in taste sensation through an unknown mechanism. Here, we describe Cl− ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from the medaka fish, currently the sole T1r amenable to structural analyses, exhibited a specific Cl− binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3, which is likely conserved across species, including human T1r3. The Cl− binding induced a conformational change in T1r2a/T1r3LBD at sub- to low-mM concentrations, similar to canonical taste substances. Furthermore, oral Cl− application to mice increased impulse frequencies of taste nerves connected to T1r-expressing taste cells and promoted their behavioral preferences attenuated by a T1r-specific blocker or T1r3 knock-out. These results suggest that the Cl− evokes taste sensations by binding to T1r, thereby serving as another preferred salt taste pathway at a low concentration.

Objective To evaluate the tolerability and efficacy of nasal pillow-noninvasive ventilation (NP-NIV) compared with high-flow nasal therapy (HFNT) in postsurgical patients. Methods This propensity score-matched retrospective study enrolled postoperative patients that received NP-NIV (NP-NIV group) or HFNT (HFNT group) in the intensive care unit. Data were collected from their medical records and the tolerability and respiratory status before and after extubation were compared between the two groups. Results The study enrolled 83 patients in the NP-NIV group and 27 patients in the HFNT group. After propensity score matching, there were 19 patients in each group. After matching, there were no significant differences in the baseline demographic and clinical characteristics before extubation. The tolerability was similar in both groups. When the NP-NIV group was compared with the HFNT group, the respiratory rate was significantly lower (median 16 [interquartile range, 14–17] versus median 19 [interquartile range, 18–26], respectively) and the partial pressure of arterial oxygen/fraction of inspired oxygen ratio was significantly higher (median 205 [174–256] versus median 155 [130–192], respectively) at 1 h after extubation. Conclusion NP-NIV was equally well tolerated and provided better respiratory support than HFNT in postsurgical patients.

Summary: The assessment of the efficacy and safety of coronavirus disease 2019 (COVID-19) vaccines in actual practice is extremely important, and monitoring efforts are being implemented worldwide. In Japan, a joint council in the Ministry of Health, Labour and Welfare is held every two to three weeks to summarise information on the adverse events following COVID-19 vaccination, with careful assessment of individual case safety reports and comparison with background incidence rates. In 2021, the joint council mainly reviewed anaphylaxis, death, myocarditis/pericarditis, and thrombosis with thrombocytopenia syndrome. These activities resulted in several safety-related regulatory actions, including the revision of vaccine package inserts with warnings about myocarditis/pericarditis. International sharing of vaccine safety information, as well as details of the evaluation systems, is important for international discussion and decision-making on better safety monitoring of COVID-19 vaccines.

Abstract We aimed to construct a novel population pharmacokinetics (PPK) model of doripenem (DRPM) for Japanese patients in intensive care unit, incorporating the clearance of DRPM by continuous renal replacement therapy (CRRT). Twenty-one patients treated with DRPM (0.25 or 0.5 g) by intravenous infusion over 1 h were included in the study. Nine of the 21 patients were receiving CRRT. Plasma samples were obtained before and 1, 2, 4, 6 and 8 h after the first DRPM administration. PPK analysis was conducted by nonlinear mixed effects modeling using a two-compartment model. Total clearance (CLtotal) in the model was divided into CRRT clearance (CLCRRT) and body clearance (CLbody). The final model was: CLtotal (L h−1) = CLbody(non-CRRT) = 3.65 × (Ccr/62.25)0.64 in the absence of CRRT, or = CLbody(CRRT) + CLCRRT = 2.49 × (Ccr/52.75)0.42 + CLCRRT in the presence of CRRT; CLCRRT = QE × 0.919 (0.919 represents non-protein binding rate of DRPM); V1 (L) = 10.04; V2 (L) = 8.13; and Q (L h−1) = 3.53. Using this model, CLtotal was lower and the distribution volumes (V1 and V2) tended to be higher compared to previous reports. Also, Ccr was selected as a significant covariate for CLbody. Furthermore, the contribution rate of CLCRRT to CLtotal was 30–40%, suggesting the importance of drug removal by CRRT. The population analysis model used in this study is a useful tool for planning DRPM regimen and administration. Our novel model may contribute greatly to proper use of DRPM in patients requiring intensive care.

Objectives: To analyse plasmids carrying blaIMP-6 in Klebsiella pneumoniae isolates obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. Methods: Plasmids harbouring blaIMP-6 were characterised by the whole-genome sequencing of four Klebsiella pneumoniae isolates carrying blaIMP-6, and compared with the pKPI-6 plasmid, which is widespread in western Japan, through pulsed-field gel electrophoresis, Southern blotting, bacterial conjugation, and qPCR. Results: Whole-genome sequencing analysis revealed that three of the four isolates carried approximately 50 kbp plasmids similar to the pKPI-6 plasmid; however, one isolate carried a 250 kbp plasmid harbouring blaIMP-6 (pE196_IMP6). So far, all of the reported plasmids carrying blaIMP-6 were similar to the pKPI-6 plasmid, and this plasmid was a novel blaIMP6-carrier. The size and transferability of this plasmid was confirmed by Southern hybridisation and conjugation experiments. It was demonstrated that the generation of plasmid pE196_IMP6 was due to an intramolecular transposition mediated by IS26, and a homologous recombination between plasmids pKPI-6 and pE013 that was obtained from another carbapenem-resistant Enterobacteriaceae isolate in this analysis. As a result of co-integration with pE013, pE196_IMP6 acquired six additional pairs of type II toxin-antitoxin systems that pKPI-6 does not carry. Transcription of all of the toxin-antitoxin systems were confirmed in an isolate carrying pE196_IMP6 by qPCR. Conclusions: This study detected a novel plasmid carrying blaIMP-6, and revealed the origin of this plasmid. Toxin-antitoxin system acquisition could enable pE196_IMP6 maintenance persistently through successions, even without selection pressure by the clinical usage of antimicrobials, generating broad dissemination and longer carbapenem-resistant Enterobacteriaceae colonisation duration in patients.

Abstract Background Neutrophil extracellular traps (NETs) are fibrous structures released from activated neutrophils. NET formation has been reported to be associated with acute respiratory distress syndrome (ARDS). However, there are no reports dealing with serial changes of NET formation in tracheal aspirate of ARDS patients. Case presentation We report three cases of ARDS. Case 1 is a 69-year-old man with necrotizing fasciitis of the buttocks, case 2 is a 49-year-old woman with extensive burns (80% of total body surface), and case 3 is a 73-year-old woman with severe bacterial pneumonia. We found abundant expression of citrullinated histone H3 (Cit-H3) and the formation of NETs at the onset of ARDS in all cases. The amounts of Cit-H3 and NETs decreased with the amelioration of respiratory failure in cases 1 and 2. In case 2, the amounts of Cit-H3 and NETs increased with aggravation of infection and respiratory status. In case 3, the abundant expression of Cit-H3 and NETs persisted; the patient did not recover from ARDS and eventually died. Cit-H3 and NETs were found in tracheal aspirates even if the patients had no direct injury to the lung as in cases 1 and 2. Conclusions In these three cases, the formation of NETs was observed in tracheal aspirate of patients with ARDS by either direct or indirect insults to the lung. The amount of NET formation changed dynamically over the clinical course of each patient.

Abstract Objective Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to public and clinical health because of their high levels of resistance to various antibiotics. We assessed the efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) against Imipenemase (IMP)-producing CRE, using the checkerboard method and time-killing assay on 13 Enterobacteriaceae isolates harboring bla IMP-1 (4 Enterobacter hormaechei, 5 Escherichia coli, and 4 Klebsiella pneumoniae isolates) and 13 isolates harboring bla IMP-6 (8 E. coli and 5 K. pneumoniae isolates). Results Minimum inhibitory concentrations (MICs) of MEM and CMZ ranged from 2 to 64 and 64 to 2048 μg/mL, respectively. Checkerboard method demonstrated the synergy of the MEM/CMZ combination in all the tested IMP-producing CRE isolates, and the time-kill assay indicated a bactericidal effect for both bla IMP-1 and bla IMP-6 positive CRE when MEM/CMZ combination was used. In vitro, the MEM/CMZ combination was potentially effective against IMP-1- or IMP-6-producing CRE. Further investigations including in vivo animal studies and clinical studies are warranted to corroborate the clinical utility of the novel combination therapy.

Abstract Background Active surveillance has the potential to prevent nosocomial transmission of carbapenem-resistant Acinetobacter baumannii (CRAB). We assessed whether rapid diagnosis using clinical specimen-direct loop-mediated isothermal amplification (LAMP), a rapid molecular diagnostic assay, and subsequent intervention, could reduce CRAB nosocomial transmission in intensive care units (ICUs). Methods A before and after (quasi-experimental) study was conducted in two ICUs at the Mahidol University Faculty of Medicine Ramathibodi Hospital with 3 months of observational period followed by 9 months of interventional period. All patients were screened for CRAB using both the culture and LAMP method from rectal swab and/or bronchial aspirates (intubated patients only) upon admission, weekly thereafter, and upon discharge. During the pre-intervention period, we performed contact precautions based on culture results. In contrast, during the intervention period, we initiated contact precautions within a few hours after sample collection on the basis of LAMP results. Results A total of 1335 patients were admitted to the ICUs, of which 866 patients (pre-intervention period: 187; intervention period: 679) were eligible for this study. Incidence rate of CRAB infection decreased to 20.9 per 1000 patient-days in the intervention period from 35.2 in the pre-intervention period (P

We demonstrated the multimerization of plasmids harboring carbapenemase genes, and multimeric plasmids of various discrete sizes existed in a host bacterial cell of Escherichia coli

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. A previous CRE surveillance study on 1,507 patients from 43 hospitals in Osaka, Japan, revealed that 12% of patients carried CRE and that 95% of the CRE isolates were IMP-type carbapenemase producers. Here, the mechanisms for this regional dissemination of a single carbapenemase gene were investigated. Since the dissemination of CRE is primarily due to the transmission of carbapenemase genes located on plasmids, we analyzed the plasmidome of 230 CRE isolates carrying blaIMP by whole-genome sequencing and Southern blotting. blaIMP-6 was found to be predominantly disseminated among chromosomally distinct isolates through the pKPI-6 plasmid. Underlying the vast clonal dissemination of pKPI-6, various subpopulations deriving from pKPI-6 were identified, which had acquired advantages for the dissemination of CRE isolates. A cluster exhibiting heteroresistance against meropenem by the transcriptional regulation of blaIMP-6 caused an outbreak likely through covert transmission of blaIMP-6. For stable carriage of blaIMP-6, they occasionally integrated blaIMP-6 on their chromosomes. In addition, we detected one isolate that broadened the range of antimicrobial resistance through a single point mutation in blaIMP-6 on pKPI-6. Multifaceted analysis of the plasmidome granted us more accurate perspectives on the horizontal spread of CRE isolates, which is difficult to trace only by comparing the whole genomes. This study revealed the predominant spread of a specific carbapenemase-encoding plasmid accompanying the emergence of phenotypically diverse derivatives, which may facilitate further dissemination of CRE in various environments. IMPORTANCE Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying blaIMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6.

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) infections continue to be a leading problem in health care facilities worldwide. Methods This single-center retrospective cohort study consisted of a derivation phase and a validation phase. The derivation phase included all patients admitted to Osaka University Hospital between May 2010 and April 2011. We proposed a provisional available, bed-sided, comprehensive (ABC) score, and evaluated its accuracy using the clinical diagnosis as a reference. We subsequently revised ABC scores based on k coefficient scores of each variable; this revision was validated by applying it to another patient population. Results A total of 172 patients and 154 cases were enrolled in the derivation and validation studies, respectively. The revised ABC score consisted of four simple variables: type of clinical specimen (1 to 3 points), Gram-staining result (1 point), presence of local inflammation (2 points), and a systemic inflammatory response (2 points). A revised score of ≥5 points was sensitive (93.8%) and specific (90.6%), and the area under the receiver-operating curve was 0.969 (95% CI; 0.957–1). Conclusions We developed a simple and comprehensive scoring system for diagnosis of nosocomial MRSA infections; this system is applicable in a wide variety of situations.