Emergence and Evolution of Unique Plasmids Harboring blaIMP-70 and blaCTX-M-253 in Multidrug-Resistant Providencia rettgeri.

Although the prevalence of carbapenem-resistant Enterobacterales remains low in Japan, these bacteria are a growing problem worldwide, owing to their multidrug resistance phenotype. We isolated a multidrug-resistant Providencia rettgeri strain, NR1418, harboring a rare blaIMP variant, blaIMP-70, a novel blaCTX-M variant, designated blaCTX-M-253, and blaMOX-1. This strain is resistant to ß-lactams, amikacin, levofloxacin, and colistin. Genomic analysis revealed that NR1418 carries two plasmids, designated pNR1418-1 and pNR1418-2. The pNR1418-1 plasmid harbors blaCTX-M-253, blaTEM-1, and blaMOX-1, while the pNR1418-2 plasmid harbors blaIMP-70, which is located in a class 1 integron. Both plasmids exhibit high similarities with the plasmid of the P. rettgeri isolate BML2526, which also harbors blaIMP-70 and was identified in the same region of Japan as NR1418 at a different point in time. This indicates the possibility of the emergence and evolution of IMP-70-producing P. rettgeri and suggests that the plasmid of BML2526 may have occurred following recombination of the two plasmids harbored by NR1418. Further, blaIMP-70 and blaCTX-M-253 were found on unique plasmids, indicating that they likely evolved through mutations and recombination. IMPORTANCE Although Providencia rettgeri is an opportunistic pathogen, its intrinsic resistance to colistin and tigecycline makes the treatment of carbapenem-resistant P. rettgeri challenging. We isolated a multidrug-resistant P. rettgeri strain which harbored a rare blaIMP variant, blaIMP-70, a novel blaCTX-M variant, blaCTX-M-253, and blaMOX-1 from a urinary sample obtained in Osaka, Japan. We investigated its genetic structure and evaluated the evolution of the plasmids carrying these genes. We show that blaIMP-70, blaCTX-M-253, and blaMOX-1 are present on unique plasmids and that they have high similarities to the plasmid of another IMP-70-producing P. rettgeri isolate that was identified as being from the same location. The evolution of plasmids through mutations and recombination may play a role in the development and spread of multidrug resistance.

Evaluation of NG-Test CARBA 5 for the detection of carbapenemase-producing Gram-negative bacilli.

Carbapenemase-producing Enterobacterales (CPE) pose one of the most serious antimicrobial resistance threats to public health worldwide. The outcome of CPE infection differs depending on the resistance mechanism. Therefore, rapid detection of CPE infection is essential for optimizing patient management. The carbapenem inactivation method (CIM) and modified CIM (mCIM) are standard methods for detecting CPE, but they usually require 24 h to generate results. Recently, an immunochromatographic assay, NG-Test CARBA 5, has become commercially available. It detects the five most common carbapenemase producers (KPC, IMP, NDM, VIM, and OXA-48) rapidly and accurately. We aimed to evaluate the diagnostic accuracy of NG-Test CARBA 5 for detecting carbapenemase-producing Gram-negative bacilli (CPGNB). We used 116 carbapenemase-producing strains and 48 non-carbapenemase-producing strains. Of the 116 carbapenemase-producing strains, 107 harboured genes for at least one of the five most common carbapenemases, KPC, IMP, NDM, VIM, and OXA-48-like. Forty-eight non-carbapenemase-producing strains, including carbapenem-resistant Enterobacterales, harboured genes for extended-spectrum ß-lactamases (CTX-M groups [n=25] and SHV groups [n=2]) or plasmid-mediated AmpC ß-lactamases (DHA [n=3], CMY-2 [n=2], and CFE-1 [n=1]). Antimicrobial susceptibility was tested using the agar dilution method, according to the Clinical and Laboratory Standards Institute guidelines. Of the 116 carbapenemase-producing strains, 79 were resistant to at least meropenem or imipenem. The sensitivity and specificity of the NG-Test CARBA 5 for the strains were 99.1 % (106 strains positive for 107 strains of the five most common carbapenemase producers) and 100 % (60 strains negative for other types of CPGNB [n=10] and non-CPGNB strains [n=48]), respectively. The carbapenemase-producing strain with a false-negative result produced IMP-66. The NG-Test CARBA 5 had high sensitivity and specificity for detecting carbapenemase-producing strains.

Impedance Characteristics of Monolayer and Bilayer Graphene Films with Biofilm Formation and Growth.

Biofilms are the result of bacterial activity. When the number of bacteria (attached to materials' surfaces) reaches a certain threshold value, then the bacteria simultaneously excrete organic polymers (EPS: extracellular polymeric substances). These sticky polymers encase and protect the bacteria. They are called biofilms and contain about 80% water. Other components of biofilm include polymeric carbon compounds such as polysaccharides and bacteria. It is well-known that biofilms cause various medical and hygiene problems. Therefore, it is important to have a sensor that can detect biofilms to solve such problems. Graphene is a single-atom-thick sheet in which carbon atoms are connected in a hexagonal shape like a honeycomb. Carbon compounds generally bond easily to graphene. Therefore, it is highly possible that graphene could serve as a sensor to monitor biofilm formation and growth. In our previous study, monolayer graphene was prepared on a glass substrate by the chemical vapor deposition (CVD) method. Its biofilm forming ability was compared with that of graphite. As a result, the CVD graphene film had the higher sensitivity for biofilm formation. However, the monolayer graphene has a mechanical disadvantage when used as a biofilm sensor. Therefore, for this new research project, we prepared bilayer graphene with high mechanical strength by using the CVD process on copper substrates. For these specimens, we measured the capacitance component of the specimens' impedance. In addition, we have included a discussion about the possibility of applying them as future sensors for monitoring biofilm formation and growth.

Stereoselective Syntheses of trans-Anhydromevalonic Acid and trans-Anhydromevalonyl Group-Containing Natural Products.

Collective total syntheses of trans-anhydromevalonic acid (tAHMA) and trans-anhydromevalonyl (tAHM) group-containing natural products (pestalotiopin A, pestalotiopamide C, pestalotiopamide D, farinomalein E, eleutherazine B, and trichocyclodipeptide A) were achieved using tAHMA esters as key intermediates. To this end, tAHMA tert-butyl ester was newly prepared by Z-vinyltosylation of tert-butyl 3-oxo-5-((triisopropylsilyl)oxy)pentanoate followed by the Negishi cross-coupling reaction with Me2Zn. tAHMA esters were converted to the target natural products via esterification or amidation. Comparison of the spectroscopic data of synthetic and natural products confirmed the E-configuration of the tAHM moieties in the natural products.

Predominance of CTX-M-9 Group Among ESBL-Producing Escherichia coli Isolated from Healthy Individuals in Japan.

The detection rate of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales, microorganisms associated with health care settings, has significantly increased worldwide. Moreover, their community incidence has increased in several countries. In this study, we investigated the prevalence and genetic diversity of ESBL-producing Escherichia coli isolated from 547 nonduplicated stool specimens from healthy Japanese individuals, between 2015 and 2019. E. coli were isolated on deoxycholate-hydrogen sulfide-lactose (DHL) agar and identified by MALDI-TOF MS, ESBL were screened through disk diffusion method (cefotaxime with or without clavulanate), and genetic detection and genotyping were performed by PCR and DNA sequencing. Clonal similarities between ESBL-producing and nonproducing isolates were assessed by multilocus sequence typing (MLST). The prevalence of ESBL-producing E. coli was 9.7% (53/547). These bacteria harbored CTX-M genes, from which CTX-M-9 (31/53, 58.5%) and CTX-M-1 (13/53, 24.5%) groups were the predominant. The MLST analysis revealed that ST131 genotype prevailed within ESBL-producing E. coli (15/53), whereas ST95 (10/53) and ST73 (8/53) prevailed among non-ESBL producers, with ST131 being present in only four isolates. Overall, a high prevalence rate of CTX-M-type ESBL-producing E. coli was detected. CTX-M-9 group-producing ST131 predominated among healthy Japanese individuals, similar to that observed in hospital isolates. CTX-M-type ESBL may disseminate clonally among hospital patients and subsequently, within the community.

Total Synthesis and Structure Confirmation of trans-Anhydromevalonate-5-phosphate, a Key Biosynthetic Intermediate of the Archaeal Mevalonate Pathway.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently, trans-anhydromevalonate-5-phosphate (tAHMP) was isolated as a new biosynthetic intermediate of the archaeal mevalonate pathway. In this study, we would like to report the first synthesis of tAHMP and its enzymatic transformation using one of the key enzymes, mevalonate-5-phosphate dehydratase from a hyperthermophilic archaeon, Aeropyrum pernix. Starting from methyl tetrolate, a Cu-catalyzed allylation provided an E-trisubstituted olefin in a stereoselective manner. The resulting E-olefin was transformed to tAHMP by cleavage of the olefin and phosphorylation. The structure of the synthetic tAHMP was unambiguously determined by NOESY analysis.

Contrast Variation Small-Angle Neutron Scattering Study of Solubilization of Perfumes in Cationic Surfactant Micelles.

Perfume solubilization is an important process in the production of commercial products such as beverages, foods, and cosmetics. In the present study, small-angle neutron scattering (SANS) experiments were performed to investigate the solubilization behavior of perfumes in cetyltrimethylammonium bromide (CTAB) micelles. The solubilization of linalool (LL) and l-menthol (MT), which are relatively hydrophilic perfumes, did not change the size of the CTAB micelles although the perfumes were incorporated in the micelles, as indicated by a decrease in scattering length density. On the other hand, the solubilization of d-limonene (LN), a hydrophobic perfume, led to the swelling of CTAB micelles. An internal contrast variation SANS study was performed by the deuteration of CTAB molecules to directly observe the perfumes in the micelles. The radius of d-CTAB micelles solubilizing LL or MT corresponds to that of h-CTAB, which indicates that these perfumes are accommodated in the palisade layers of the micelles and are homogeneously distributed in the micelles. On the other hand, LN formed small droplets, as indicated by the SANS profile, which implies the solubilization of LN molecules in the core of the CTAB micelles. We found that the relatively hydrophilic perfumes (LL and MT) show less impact on the sizes of the cationic micelles in comparison to nonionic micelles. Thus, the internal contrast variation method of SANS allowed the direct observation of the solubilization sites of perfumes with different hydrophilicity-hydrophobicity balances. This method is a powerful tool to determine the solubilization states that affect the solubilization capacity, volatilization, or release speed of perfumes.

Predicting 72-h mortality in patients with extremely high random plasma glucose levels: A case-controlled cross-sectional study.

ABSTRACT: The risk factors associated with 72-hours mortality in patients with extremely high levels of random plasma glucose (RPG) remain unclear.To explore the risk factors predictive of 72-hours mortality in patients with extremely high RPG under heterogenos pathophysiological conditions.Retrospective, single-center, case-controlled cross-sectional study.University teaching hospital.Adults over age 18 were selected from the medical records of patients at the Saitama Medical Center, Japan, from 2004 to 2013.Extremely high RPG (≥500 mg/dl).Mortality at 72 hours following the RPG test, regardless of hospitalization or in an outpatient setting. Multivariate logistic regression analysis was performed with adjustment for age, sex, body mass index (BMI), and RPG level. The final prediction model was built using the logistic regression model with a higher C-statistic, specificity, and sensitivity.A total of 351 patients with RPG ≥500 mg/dl were identified within the 10-year period. The 72-hours mortality rate was 16/351 (4.6%). The C-statistics of the 72-hours mortality prediction model with serum albumin (ALB) and creatine kinase (CK) was 0.856. The probability of 72-hours mortality was calculated as follows: 1/[1 + exp (-5.142 + 0.901log (CK) -1.087 (ALB) + 0.293 (presence (1) or absence (0) of metastatic solid tumor)]. The sensitivity and specificity of this model was 75.5%.The independent risk factors associated with 72-hours mortality in patients with RPG ≥500 mg/dl are hypoalbuminemia, elevated CK, and presence of a metastatic solid tumour. Further research is needed to understand the mechanisms and possible interventions to prevent mortality associated with extremely high RPG.

Prediction of 72-hour mortality in patients with extremely high serum C-reactive protein levels using a novel weighted average of risk scores.

The risk factors associated with mortality in patients with extremely high serum C-reactive protein (CRP) levels are controversial. In this retrospective single-center cross-sectional study, the clinical and laboratory data of patients with CRP levels ≥40 mg/dL treated in Saitama Medical Center, Japan from 2004 to 2017 were retrieved from medical records. The primary outcome was defined as 72-hour mortality after the final CRP test. Forty-four mortal cases were identified from the 275 enrolled cases. Multivariate logistic regression analysis (MLRA) was performed to explore the parameters relevant for predicting mortality. As an alternative method of prediction, we devised a novel risk predictor, "weighted average of risk scores" (WARS). WARS features the following: (1) selection of candidate risk variables for 72-hour mortality by univariate analyses, (2) determination of C-statistics and cutoff value for each variable in predicting mortality, (3) 0-1 scoring of each risk variable at the cutoff value, and (4) calculation of WARS by weighted addition of the scores with weights assigned according to the C-statistic of each variable. MLRA revealed four risk variables associated with 72-hour mortality-age, albumin, inorganic phosphate, and cardiovascular disease-with a predictability of 0.829 in C-statistics. However, validation by repeated resampling of the 275 records showed that a set of predictive variables selected by MLRA fluctuated occasionally because of the presence of closely associated risk variables and missing data regarding some variables. WARS attained a comparable level of predictability (0.837) by combining the scores for 10 risk variables, including age, albumin, electrolytes, urea, lactate dehydrogenase, and fibrinogen. Several mutually related risk variables are relevant in predicting 72-hour mortality in patients with extremely high CRP levels. Compared to conventional MLRA, WARS exhibited a favorable performance with flexible coverage of many risk variables while allowing for missing data.

A retrospective cross-sectional study for predicting 72-h mortality in patients with serum aspartate aminotransferase levels ≥ 3000 U/L.

Risk factors associated with 72-h mortality in patients with extremely high serum aspartate aminotransferase levels (AST; ≥ 3000 U/L) are unknown. This single-centre, retrospective, case-controlled, cross-sectional study obtained data from medical records of adult patients treated at Saitama Medical Center, Japan, from 2005 to 2019. We conducted a multivariate logistic after adjusting for age, sex, height, weight, body mass index, Brinkman Index, vital signs, biochemical values, updated Charlson Comorbidity Index (CCI) score, CCI components, and underlying causes. A logistic regression model with selected validity risks and higher C-statistic for predicting 72-h mortality was established. During the 15-year period, 428 patients (133 non-survivors and 295 survivors [cases and controls by survival < 72 and ≥ 72 h, respectively]) with AST levels ≥ 3000 U/L were identified. The 72-h mortality rate was 133/428 (31.1%). The model used for predicting 72-h mortality through the assessment of alkaline phosphatase, creatine kinase, serum sodium, potassium, and phosphorus levels had a C-statistic value of 0.852 (sensitivity and specificity, 76.6%). The main independent risk factors associated with 72-h mortality among patients with AST levels ≥ 3000 U/L included higher serum values of alkaline phosphatase, creatine kinase, serum sodium, potassium, and phosphorus.

The Role of nmcR, ampR, and ampD in the Regulation of the Class A Carbapenemase NmcA in Enterobacter ludwigii.

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all ß-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that bla NmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of ß-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent ß-lactamase inducer. In contrast, carbapenems induced ß-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in ß-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery.

Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.

Synthesis of 5-Aryl-3(2 H)-furanones Using Intramolecular Cyclization of Sulfonium Salts.

Base-induced intramolecular cyclization of novel (4-aryl-2,4-dioxobutyl)methylphenylsulfonium salts prepared from the commercially available 1-arylethanone by a cost-effective process is described in this paper. The reaction was completed within 10 min to produce a family of 2-unsubstituted 5-aryl-3(2 H)-furanones in excellent yield. This procedure is simple, and can be carried out under mild conditions and an ambient atmosphere.

Combinatorial Biosynthesis of (+)-Daurichromenic Acid and Its Halogenated Analogue.

Daurichromenic acid is a meroterpenoid with various pharmacological activities that is biosynthesized from grifolic acid in Rhododendron dauricum. Heterologous expression of grifolic acid synthases from Stachybotrys bisbyi and a daurichromenic acid synthase from R. dauricum in Aspergillus oryzae mediated three-step combinatorial biosynthesis of (+)-daurichromenic acid through enantioselective 6-endo-trig cyclization. Additional introduction of a halogenase from Fusarium sp. into the strain resulted in the biosynthesis of (+)-5-chlorodaurichromenic acid, which exceeds the antibacterial activity of the original compounds.