Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Functional Divergence and Origin of the Vertebrate Praja Family.

The Praja family is an E3 ubiquitin ligase, promoting polyubiquitination and subsequent degradation of substrates. It comprises two paralogs, praja1 and praja2. Prior research suggests these paralogs have undergone functional divergence, with examples, such as their distinct roles in neurite outgrowth. However, the specific evolutionary trajectories of each paralog remain largely unexplored preventing mechanistic understanding of functional differences between paralogs. Here, we investigated the phylogeny and divergence of the vertebrate Praja family through molecular evolutionary analysis. Phylogenetic examination of the vertebrate praja revealed that praja1 and praja2 originated from the common ancestor of placentals via gene duplication, with praja1 evolving at twice the rate of praja2 shortly after the duplication. Moreover, a unique evolutionary trajectory for praja1 relative to other vertebrate Praja was indicated, as evidenced by principal component analysis on GC content, codon usage frequency, and amino acid composition. Subsequent motif/domain comparison revealed conserved N terminus and C terminus in praja1 and praja2, together with praja1-specific motifs, including nuclear localization signal and Ala-Gly-Ser repeats. The nuclear localization signal was demonstrated to be functional in human neuroblastoma SH-SY5Y cells using deletion mutant, while praja2 was exclusively expressed in the nucleus. These discoveries contribute to a more comprehensive understanding of the Praja family's phylogeny and suggest a functional divergence between praja1 and praja2. Specifically, the shift of praja1 into the nucleus implies the degradation of novel substrates located in the nucleus as an evolutionary consequence.

Phylogenetic tree reconstruction via graph cut presented using a quantum-inspired computer.

Phylogenetic trees are essential tools in evolutionary biology that present information on evolutionary events among organisms and molecules. From a dataset of n sequences, a phylogenetic tree of (2n-5)!! possible topologies exists, and determining the optimum topology using brute force is infeasible. Recently, a recursive graph cut on a graph-represented-similarity matrix has proven accurate in reconstructing a phylogenetic tree containing distantly related sequences. However, identifying the optimum graph cut is challenging, and approximate solutions are currently utilized. Here, a phylogenetic tree was reconstructed with an improved graph cut using a quantum-inspired computer, the Fujitsu Digital Annealer (DA), and the algorithm was named the "Normalized-Minimum cut by Digital Annealer (NMcutDA) method". First, a criterion for the graph cut, the normalized cut value, was compared with existing clustering methods. Based on the cut, we verified that the simulated phylogenetic tree could be reconstructed with the highest accuracy when sequences were diverged. Moreover, for some actual data from the structure-based protein classification database, only NMcutDA could cluster sequences into correct superfamilies. Conclusively, NMcutDA reconstructed better phylogenetic trees than those using other methods by optimizing the graph cut. We anticipate that when the diversity of sequences is sufficiently high, NMcutDA can be utilized with high efficiency.

Mutational and functional analysis of Glucose transporter I deficiency syndrome.

OBJECTIVE: We investigated a correlation between a mutation in the SLC2A1 gene and functional disorders in Glucose transporter I deficiency syndrome (GLUT1DS). METHODS: We performed direct sequence analysis of SLC2A1 in a severe GLUT1DS patient and identified a novel frame shift mutation, c.906_907insG, p.V303fs. We created a plasmid vector carrying the c.906_907insG mutation, as well as A405D or R333W in the SLC2A1, which are found in patients with mild and moderate GLUT1DS severity, respectively. We transiently expressed these mutants and wild type SLC2A1 plasmids in a human embryonic kidney cell line (HEK293), and performed immunoblotting, immunofluorescence, and enzymatic photometric 2-deoxyglucose (2DG) uptake assays. RESULTS: GLUT1 was not detected after transient expression of the SLC2A1 plasmid carrying c.906_907insG by either immunoblotting or immunofluorescence. The degree of glucose transport reduction as determined by enzymatic photometric 2DG assay uptake correlated with disease severity. CONCLUSIONS: Enzymatic photometric 2DG uptake study appears to be a suitable functional assay to predict the effect of SLC2A1 mutations on GLUT1 transport.

Synthetic aporphine alkaloids are potential therapeutics for Leigh syndrome.

Mitochondrial diseases are mainly caused by dysfunction of mitochondrial respiratory chain complexes and have a variety of genetic variants or phenotypes. There are only a few approved treatments, and fundamental therapies are yet to be developed. Leigh syndrome (LS) is the most severe type of progressive encephalopathy. We previously reported that apomorphine, an anti- "off" agent for Parkinson's disease, has cell-protective activity in patient-derived skin fibroblasts in addition to strong dopamine agonist effect. We obtained 26 apomorphine analogs, synthesized 20 apomorphine derivatives, and determined their anti-cell death effect, dopamine agonist activity, and effects on the mitochondrial function. We found three novel apomorphine derivatives with an active hydroxy group at position 11 of the aporphine framework, with a high anti-cell death effect without emetic dopamine agonist activity. These synthetic aporphine alkaloids are potent therapeutics for mitochondrial diseases without emetic side effects and have the potential to overcome the low bioavailability of apomorphine. Moreover, they have high anti-ferroptotic activity and therefore have potential as a therapeutic agent for diseases related to ferroptosis.

Apomorphine is a potent inhibitor of ferroptosis independent of dopaminergic receptors.

Originally, apomorphine was a broad-spectrum dopamine agonist with an affinity for all subtypes of the Dopamine D1 receptor to the D5 receptor. We previously identified apomorphine as a potential therapeutic agent for mitochondrial diseases by screening a chemical library of fibroblasts from patients with mitochondrial diseases. In this study, we showed that apomorphine prevented ferroptosis in fibroblasts from various types of mitochondrial diseases as well as in normal controls. Well-known biomarkers of ferroptosis include protein markers such as prostaglandin endoperoxide synthase 2 (PTGS2), a key gene for ferroptosis-related inflammation PTGS2, lipid peroxidation, and reactive oxygen species. Our findings that apomorphine induced significant downregulation of PTSG2 and suppressed lipid peroxide to the same extent as other inhibitors of ferroptosis also indicate that apomorphine suppresses ferroptosis. To our knowledge, this is the first study to report that the anti-ferroptosis effect of apomorphine is not related to dopamine receptor agonist action and that apomorphine is a potent inhibitor of ferroptotic cell death independent of dopaminergic receptors.

Establishment of a flow cytometry screening method for patients with glucose transporter 1 deficiency syndrome.

Objective: We assessed the usefulness of flow cytometry as a functional assay to measure glucose transporter 1 (GLUT1) levels on the surface of red blood cells (RBCs) from Japanese patients with glucose transporter 1 deficiency syndrome (Glut1DS). Methods: We recruited 13 genetically confirmed Glut1DS patients with a solute carrier family 2 member 1 (SLC2A1) mutation (eight missense, one frameshift, two nonsense, and two deletion) and one clinically suspected Glut1DS-like patient without an SLC2A1 mutation, and collected whole blood with informed consent. We stained pelleted RBCs (1 µL) from the patients with a Glut1.RBD ligand and anti-glycophorin A antibody, which recognizes a human RBC membrane protein, and analyzed the cells using flow cytometry. Results: Relative GLUT1 levels quantified by flow cytometry in 11 of 13 patients with definite Glut1DS were 90% below those of healthy controls. Relative GLUT1 levels were not reduced in two of 13 Glut1DS patients who had a missense mutation and no intellectual disability and one Glut1DS-like patient without an SLC2A1 mutation. Relative GLUT1 levels were significantly reduced in Glut1DS patients with an SLC2A1 mutation, more severe intellectual disability, and spasticity. Conclusions: This method to detect GLUT1 levels on RBCs is simple and appears to be an appropriate screening assay to identify severe Glut1DS patients in the early stage before the development of irreversible neurologic damage caused by chronic hypoglycorrhachia.

Data on the solution and processing time reached when constructing a phylogenetic tree using a quantum-inspired computer.

Phylogenetic trees provide insight into the evolutionary trajectories of species and molecules. However, because (2n-5)! Phylogenetic trees can be constructed from a dataset containing n sequences, but this method of phylogenetic tree construction is not ideal from the viewpoint of a combinatorial explosion to determine the optimal tree using brute force. Therefore, we developed a method for constructing a phylogenetic tree using a Fujitsu Digital Annealer, a quantum-inspired computer that solves combinatorial optimization problems at a high speed. Specifically, phylogenetic trees are generated by repeating the process of partitioning a set of sequences into two parts (i.e., the graph-cut problem). Here, the optimality of the solution (normalized cut value) obtained by the proposed method was compared with the existing methods using simulated and real data. The simulation dataset contained 32-3200 sequences, and the average branch length according to a normal distribution or the Yule model ranged from 0.125 to 0.750, covering a wide range of sequence diversity. In addition, the statistical information of the dataset is described in terms of two indices: transitivity and average p-distance. As phylogenetic tree construction methods are expected to continue to improve, we believe that this dataset can be used as a reference for comparison and confirmation of the validity of the results. Further interpretation of these analyses is explained in W. Onodera, N. Hara, S. Aoki, T. Asahi, N. Sawamura, Phylogenetic tree reconstruction via graph cut presented using a quantum-inspired computer, Mol. Phylogenet. Evol. 178 (2023) 107636.

[A case of vesical endometriosis that worsened during the early pregnancy period].

We report a case of vesical endometriosis that worsened during the early pregnancy period. A 37-year old woman had been under treatment for endometriosis (including vesical endometriosis) by a gynecologist during the past 10 years. She was treated for sterility 1 year ago, and became pregnant through in vitro fertilization. In her 8th gestational week, she complained of gross hematuria at our hospital. Cystoscopic findings revealed some tumors that appeared worse than the last findings two years ago. In order to deny malignancy, transurethral resection of the bladder tumor was performed in her 12th gestational week. The pathologic diagnosis was endometriosis. She was able to stay pregnant, and delivered a girl. After delivery, cystoscopic findings revealed reduction of tumors. In most cases pregnancy cures endometriosis ; however, in this case symptoms became worse during the early stage of pregnancy. The reason for this contrary event is discussed.

Antimicrobial Properties of a Copper/Silicone Composite Membrane Prepared Using a Two-Step Immersion Process in Iodine and Copper Sulfate Solutions.

Silicone (polydimethylsiloxane) materials are widely used in various applications. Due to microbe adherence and biofilm formation at the surface of silicone materials, silicone materials must possess antibacterial properties. To achieve this, we prepared copper (Cu)−silicone composite membranes using a simple two-step process of immersion in iodine and copper sulfate solutions. Subsequent scanning electron microscopy revealed Cu nanoparticles (CuNPs) of 10 to 200 nanometers in diameter on the silicone membrane surface, which were identified as copper iodide using energy-dispersive X-ray spectroscopy. The mechanical strength of the material did not change significantly as a result of the two-step immersion treatment and the Cu/silicone membrane showed excellent antibacterial efficacy against Escherichia coli and Staphylococcus aureus, maintaining R > 2 even after a physical impact such as stomacher treatment. Additionally, the Cu ions eluted from the Cu/silicone membrane remained at very low concentrations, suggesting firm immobilization of CuNPs on the silicone membrane. This proposed antimicrobial treatment method does not require special equipment, can be performed at room temperature, and has the potential for use on silicone materials other than membranes.

[Efficacy and safety of imidafenacin in female patients with urge and mixed urinary incontinence].

The effect of imidafenacin for the treatment of overactive bladder (OAB), in female patients with urge and mixed urinary incontinence was examined. Prior to administration and at 1, 2, 3, 4, 6, 8, 10 and 12 weeks after administration, symptoms and quality of life were assessed using the overactive bladder symptom score (OABSS) and the international consultation on incontinence questionnaire-short form (ICIQ-SF), respectively. After administration, OABSS and ICIQ-SF scores were improved significantly when compared to baseline values. The incidence of adverse events was 7. 9% and none were serious. Imidafenacin was effective in female patients with urge and mixed urinary incontinence. In addition, imidafenacin rapidly improved incontinence one week after administration.

Valine metabolites analysis in ECHS1 deficiency.

Short-chain enoyl-CoA hydratase (ECHS1) is involved in amino acid and fatty acid catabolism in mitochondria and its deficiency causes Leigh syndrome or exercise-induced dystonia. More than 60 patients with this condition have been reported till date. The accumulation of intermediate metabolites of valine is assumed to be responsible for the cytotoxicity. Since protein restriction, including valine reportedly improves neurological symptoms, it is essential to consider the possible incidence of and diagnose ECHS1 syndrome in the earlier stages. This study reported the liquid chromatography with tandem mass spectrometry (LC-MS/MS) urine and plasma metabolite analysis in six cases, including four new cases with ECHS1 deficiency. The values of urine cysteine/cysteamine conjugates from valine metabolites, S-(2-carboxypropyl) cysteine/cysteamine from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine/cysteamine from acryloyl-CoA were separated between six patients and six normal controls. The LC-MS/MS analysis revealed that these metabolites can be used for the early diagnosis and evaluation of diet therapy.

Drug screening for Pelizaeus-Merzbacher disease by quantifying the total levels and membrane localization of PLP1.

BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is caused by point mutations or copy number changes in the proteolipid protein 1 gene (PLP1). PLP1 is exclusively localized in the myelin sheath of oligodendrocytes. Amino acid-substituted PLP1 protein is unable to fold properly and is subsequently degraded and/or restrictedly translated, resulting in a decrease in the PLP1 protein level and a failure to localize to the membrane. Furthermore, misfolded proteins increase the burden on the intracellular quality control system and trafficking, finally resulting in cell apoptosis. The objective of this study was to identify therapeutic chemicals for PMD by quantifying the total levels and membrane localization of PLP1. METHOD: We established a cell line stably expressing PLP1A243V fused with green fluorescent protein in oligodendrocyte-derived MO3.13 cells. We screened a chemical library composed of drugs approved for central nervous system disorders that increased both the total intensity of PLP1A243V in the whole cell and the cell membrane localization. We analyzed the change in the endoplasmic reticulum (ER) stress and the gene expression of candidate chemicals using a micro-array analysis. Finally, we tested the in vivo effectiveness using myelin synthesis deficient (msd) mice with Plp A243V . RESULTS AND CONCLUSION: Piracetam significantly increased the PLP1A243V intensity and membrane localization and decreased the ER stress. It was also shown to reverse the gene expression changes induced by PLP1A243V in a micro-array analysis. However, in vivo treatment of piracetam did not improve the survival of msd mice (Plp1A243V).

Purification of the functional plant membrane channel KAT1.

The inward-rectifying K(+) channel KAT1 is expressed mainly in Arabidopsis thaliana guard cells. The purification of functional KAT1 has never been reported. We investigated the extraction of the plant K(+) channel KAT1 with different detergents, as an example for how to select detergents for purifying a eukaryotic membrane protein. A KAT1-GFP fusion protein was used to screen a library of 46 detergents for the effective solubilization of intact KAT1. Then, a "test set" of three detergents was picked for further analysis, based on their biochemical characteristics and availability. The combination use of the selected detergents enabled the effective purification of functional KAT1 with affinity and gel-filtration chromatography.

[Detection methods and changes in metallo-beta-lactamase-producing Gram-negative rod isolates at Nagasaki University Hospital from 1991 to 2005].

Since plasmid-mediated metallo-beta-lactamase (MBL)-producing Pseudimonas aeruginosa was reported in Japan, MBL-producing Gram-negative rods (GNRs) have emerged worldwide. We developed a way to detect MBL-producing GNRs in routine examination using broth microdilution with the MBL inhibitor sodium mercaptoacetate (SMA) in frozen plates. Between 1996 and 2005, we evaluated this and other methods, including broth microdilution with another MBL inhibitor dipicolinic acid (DPA) in dry plates, conventional PCR, and a combined simple DNA preparation and enzymatic PCR product detection. The combined method is suitable for detecting IMP-type MBL-producing GNRs from numerous isolates. Broth microdilution with SMA at a concentration of 400 microg/mL had high performance and detected most PCR-positive MBL-producing GNRs in routine antimicrobial susceptibility testing. DPA in dry plates at 400 microg/mL yielded false positive results in 11.4% of isolates but worked satisfactorily at 175 microg/mL and 400 microg/mL of SMA in frozen plates. Until 1996, MBL had been detected from only 6 bacterial species, i.e., Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Achromobacter xylosoxidans, Serratia marcescens, and Citrobacter freundii, MBL-producing GNRs were later found in other glucose nonfermenting GNRs such as Acinetobacter baumanii, Acinetobacter lwoffii, and Burkholderia cepacia complex and Enterobacteriaceae. Most MBL-producing bacteria were multidrug resistant and no antimicrobial agents exist that are active against such isolates in monotherapy, making their rapid detection very important in controlling infection control.

Antibacterial Properties of Silicone Membranes after a Simple Two-Step Immersion Process in Iodine and Silver Nitrate Solutions.

　Silicone is widely used in packing materials, medical equipment, and separation membranes. Since microbial cells easily adhere to the surface of silicone materials and form biofilms, techniques for incorporating antimicrobial activity into silicone materials are in high demand. This study describes the preparation of silver (Ag)/silicone composite membranes through a simple two-step immersion process, utilizing an iodine solution followed by a silver nitrate solution at room temperature. Scanning electron microscopy (SEM) observations revealed that particles with sizes of several nanometers to several tens of nanometers were present on the silicone membrane surface; these particles were identified as silver iodide using energy-dispersive X-ray spectroscopy (EDS) . The Ag/silicone membrane possessed excellent antibacterial efficacy against Escherichia coli and Staphylococcus aureus, and the antibacterial efficacy (R) against both types of bacteria was R > 4, even after stomacher treatment or acidic treatment of pH 2-6 for 24 h. The mechanical strength of the silicone membrane was also maintained after antibacterial treatment, with Young's modulus values of 7.9±1.2 MPa and 8.3±1.5 MPa for the untreated membrane and Ag/silicone membrane, respectively (p > 0.05) . In addition, the reduction in permeation performance of the Ag/silicone membrane was only 20%, despite the antibacterial treatment on the membrane surface. This antibacterial treatment method of silicone membranes can be conducted at room temperature (25℃) without special equipment, and may be applied to other types of silicone materials.

Gene therapy for a mouse model of glucose transporter-1 deficiency syndrome.

OBJECTIVE: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene was expressed under the synapsin I promoter (AAV-hSLC2A1) and examined if AAV-hSLC2A1 administration can lead to functional improvement in GLUT1-deficient mice. METHODS: AAV-hSLC2A1 was injected into heterozygous knock-out murine Glut1 (GLUT1+/-) mice intraperitoneally (systemic; 1.85 × 1011 vg/mouse) or intra-cerebroventricularly (local; 1.85 × 1010 vg/mouse). We analyzed GLUT1 mRNA and protein expression, motor function using rota-rod and footprint tests, and blood and cerebrospinal fluid (CSF) glucose levels. RESULTS: Vector-derived RNA was detected in the cerebrum for both injection routes. In the intra-cerebroventricular injection group, exogenous GLUT1 protein was strongly expressed in the cerebral cortex and hippocampus near the injection site. In the intraperitoneal injection group, exogenous GLUT1 protein was mildly expressed in neural cells throughout the entire central nervous system. The motor function test and CSF/blood glucose ratio were significantly improved following intra-cerebroventricular injection. CONCLUSIONS: AAV-hSLC2A1 administration produced exogenous GLUT1 in neural cells and improved CSF glucose levels and motor function of heterozygous knock-out murine Glut1 mice.

Oral administration of the Aureobasidium pullulans-derived ß-glucan effectively prevents the development of high fat diet-induced fatty liver in mice.

Aureobasidium pullulans-derived ß-glucan (AP-PG) consisting of a ß-(1,3)-linked glucose main chain and ß-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD).

Stimulation of macrophages with the ß-glucan produced by aureobasidium pullulans promotes the secretion of tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

A ß-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a ß-(1,3)-linked main chain with ß-(1,6)-linked glucose side residues. Various ß-glucans consisting of ß-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial ß-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Clinical and bacteriological characteristics of IMP-type metallo-beta-lactamase-producing Pseudomonas aeruginosa.

IMP-type metallo-beta-lactamase-producing bacteria have recently emerged worldwide. We conducted a case-control study in which 69 inpatients harboring bla(IMP)-positive Pseudomonas aeruginosa and 247 control subjects with bla(IMP)-negative pathogens were investigated. Prolonged hospitalization, antineoplastic chemotherapy, corticosteroid therapy (P=.001), and indwelling urinary catheters (P=.04) were risk factors for isolation of bla(IMP)-positive pathogens. The predominant source was urine (P=.001). The duration of antibiotic treatment and the total dose (including of carbapenems) were significantly greater among case patients than among control subjects (P<.01). bla(IMP)-positive P. aeruginosa isolates were more frequently resistant to multiple drugs (P=.001) and caused more infections (P=.001) than bla(IMP)-negative pathogens. There were no significant differences in bacteriological outcome (P=.94); however, infection-related death was more frequent among case patients than among control subjects (P=.023). These results suggest that precautionary measures against the spread of bla(IMP)-positive isolates are needed, because, for most of such pathogens, no antibiotic is potent enough to be used as a single agent in treatment of infection.