Quinolones are useful synthetic antimicrobial agents in the clinical settings with broad specrum, potent efficacy, good bioavailability, and excellent safety. Therefore, development and increase of ...quinolone-resistance among clinically isolated pathogens are a worrisome problem. Recently, increasing numbers of plasmid-mediated quinolone resistance (PMQR) among clinical isolates of Enterobacteriaceae have been reported. PMQR consists of Qnr family as a protective determinant of bacterial drug-target enzymes, Aac (6')-Ib-cr as a drug-modifying enzyme, and plasmid-mediated quinolone efflux pumps. In this review, biological features of PMQR determinants including discovery, phylogeny and presumable physiological role, as well as their clinical contribution of the acquisition and development of quinolone resistance, are discussed.
ABSTRACT
Analysis of five CTX-M-2-producing
Proteus mirabilis
isolates in Japan demonstrated that
bla
CTX-M-2
was located on the chromosome in four isolates and on IncT plasmids in three isolates, ...including two isolates that also carried the gene on the chromosome. In all four isolates with chromosomal
bla
CTX-M-2
, IS
Ecp1
was responsible for the integration of the gene into the chromosome. Three different sites in the
P. mirabilis
genomic sequence were utilized as integration sites.
We investigate the evolving molecular epidemiology of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan from ...2004 to 2006.
MBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. bla IMP-1 group was the most predominant (38 isolates, 80%), followed by 3 bla IMP-7, 2 bla IMP-11 group, and 1 bla VIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole bla IMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with bla IMP-11 group or ST235 with bla IMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp.
Our data give an important "snapshot" of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background. Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by anti-platelet autoantibodies. The underlying mechanism is thought to involve the production of IgG ...autoantibodies specific for platelet membrane antigens such as glycoprotein (GP) IIb/IIIa and GPIb/IX, although the anti-platelet autoantibody testing is less sensitive. The ASH and the IWG guidelines for the management of ITP do not recommend routine testing of anti-platelet autoantibodies for the diagnosis of ITP, and thus diagnostic biomarkers for ITP remain to be developed. Although the principal pathophysiology of ITP is believed to be an IgG-mediated autoimmune disease, there are few evidence for the B-cell receptor (BCR) repertoires which are associated or related to this disorder.
Objectives. In order to identify the characteristics of IgG-BCRs that could be a potential candidate for a biomarker for primary ITP, we have investigated the comprehensive and precise repertoires of IgG-BCRs of peripheral blood B cells by using the next-generation BCR repertoire analysis comprised of high-throughput DNA sequencing and bioinformatics.
Materials and Methods. Eleven adult ITP patients and nine volunteer donors without any hematologic disorders were included in this study. Diagnostic criteria for ITP were based on the ASH and the IWG guidelines and secondary ITP was excluded. Written informed consent was obtained before drawing blood samples. Heparinized peripheral blood was taken and mononuclear cells were isolated by density gradient centrifugation. Total RNA extraction and cDNA synthesis were done by standard protocols. Unbiased amplifications of IgG-BCRs were performed by adaptor-ligation PCR and the amplicons were sequenced by next-generation sequencing. Each sequence read was analyzed by the bioinformatics software created by Repertoire Genesis Incorporation (Ibaraki, Japan), and the usage of IGHV, IGHD, IGHJ and IGHC and CDR3 sequences were determined, according to the previously reported (Iwamoto M, et al. ASH 2016, #4542). This study was approved by the institutional review board.
Results. Total 2,009,943 in-frame and 315,469 unique reads were obtained from twenty blood samples, and 29,049 to 160, 013 reads (100,497 reads in average) from each sample. Diversity of IgG BCRs was not different between the ITP patients and controls based on inverse Simpson and evenness Pielou's indexes. Comparisons of the IGHV repertoires covering fifty-nine IGHV subfamilies between the patients and controls revealed an increased usage of IGHV4-28 (0.053% vs. 0.005%, p=0.006) and a less usage of the IGHV3-15 (1.28% vs. 3.63%, p=0.04) segment in ITP patients (Fig. 1). The 220 distinct IGHV4-28-carrying sequences were identified in ITP patients and 135 out of these clones used the IGHJ4 gene segment (Fig. 2). These IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Only eight B-cell clones bearing IGHV4-28/IGHJ4 were identified in control donors. However, no identical IGHV4-28/IGHJ4-bearing B-cell clones were found across different ITP patients.
Discussion and Conclusion. The present study has demonstrated the preferential usage of the rearranged IGHV4-28/IGHJ4 gene by circulating IgG B cells in ITP. Roark et al. have previously reported the usage of IGHV3-30 by the anti-platelet immunoglobulin from two patients with ITP, demonstrated by the Fab/phage display libraries (Blood 2002;100:1388). Discordant results may be explained by the limited numbers of the patients examined or different HLA backgrounds. Although the antigen specificity of BCRs encoded by the IGHV4-28/IGHJ4 gene in the present patient cohort remains to be elucidated, our findings may contribute to the development of genetic testing for the diagnosis of ITP.
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Kitaura:Repertoire Genesis Incorporation: Employment. Suzuki:Repertoire Genesis Incorporation: Equity Ownership.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Retinoic acids showed up-regulation of ICAM-1 mRNA and cell surface expression.•TTNPB, a retinoic acid receptor agonist induced ICAM-1 expression.•HX531, retinoic acid receptor antagonist inhibited ...the effect of retinoic acids.•ICAM-1 did not contribute to the retinoic acid-induced cell survival.
Active metabolites of vitamin A, retinoic acids (RAs), are known to play critical roles in mucosal immune responses and dramatically inhibit human eosinophil apoptosis, but the detailed mechanisms have not been elucidated. We previously screened for ICAM-1 (CD54) upregulation in RA-stimulated human eosinophils by gene microarray analysis. As ICAM-1 induction and activation were observed to have a role in maintenance of eosinophil survival, we tested the hypothesis that RAs prolong eosinophil survival through ICAM-1 outside-in signaling. Blood-derived isolated eosinophils cultured with 9-cis RA and all-trans RA showed significant upregulation of ICAM-1 mRNA and cell surface expression. TTNPB, a retinoic acid receptor agonist, also induced ICAM-1 expression, while HX630, a retinoid X receptor agonist, did not. Furthermore, an RAR antagonist, HX531, completely inhibited the effect of RAs. Upregulated ICAM-1 was associated with altered kinetics of Akt, ERK, and p38 MAP kinase phosphorylation through ICAM-1 cross-linking, but an ICAM-1-blocking antibody did not affect RA-mediated cell survival. These findings indicate that RAs induce functional ICAM-1 expression through RARs, but the induced ICAM-1 does not contribute to prolongation of eosinophil survival.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Background. Idiopathic pure red cell aplasia (PRCA) and secondary PRCA not responding to the treatment of the underlying diseases are generally thought be immune-mediated and treated by ...immunosuppressive therapy. We previously conducted the PRCA2004/2006 study and reported that poor response to induction therapy and relapse of anemia were associated with death. Principal causes of death were infections and organ failure. Based on the literatures, idiopathic PRCA may represents the prodrome to myelodysplastic syndromes. Theoretically, there are two potential mechanisms of unresponsiveness to immunosuppression; the clonal hematopoiesis by the stem/progenitor cells that have undergone somatic mutations during disease progression of PRCA and the clonal changes of auto-aggressive lymphocytes reacting against erythroid progenitors.
Objectives. In this study, we investigated the somatic mutations of myeloid malignancy-associated genes in acquired PRCA in order to determine how often clonal hematopoiesis is detected in this disorder.
Materials and Methods. This study included 23 patients with chronic acquired PRCA (12 idiopathic, 7 thymoma-, 2 LGL leukemia- and 2 systemic lupus erythematosus-associated PRCA) with a median age of 62 (range: 40-62). Disease status was varying. After obtaining informed consent, heparinized blood was drawn and mononuclear cells were separated by density gradient centrifugation. Extracted genomic DNA samples were subjected to targeted sequencing for 54 myeloid malignancy-associated genes using a TruSight Myeloid Sequencing Panel kit according to the manufacturer's instruction (Illumina). Criteria for the significant somatic mutations of myeloid malignancy-associated genes in the present study were as follows: potential functional consequences such as missense, nonsense or frameshift mutations; exclusion of previously reported SNPs; being recurrently detected in two sequencing runs; variant allele frequency (VAF) exceeding 0.02 and less than 0.40. The institutional review board approved the experimental protocol.
Results. We detected some mutations of the targeted genes in 20 out of 23 patients, and the somatic mutations defined by the criteria mentioned above were found in 10 patients including 6 idiopathic, 3 thymoma-associated and one LGL leukemia-associated PRCA (Fig. 1). These 10 patients had 38 distinct mutations in 20 genes. Variant allele frequencies were 0.02 to 0.37 (median, 0.04; average, 0.06, Fig. 2). Four patients had more than one mutated genes and multiple genes were mutated in some patients (Fig. 1). The most frequently mutated gene was CUX1 that was found in four patients, and STAG2, DNMT3A, KDM6A, SMC3A, ASXL1, TET2 and TP53 were mutated in more than one patient.
Discussion/Conclusion. This study demonstrated that myeloid malignancy-associated genes were somatically mutated in 43% of acquired chronic PRCA patients. This figure appears to exceed the prevalence rate of clonal hematopoiesis of indeterminate potential (CHIP) in the general population with the age of 60s. These mutations were presumably carried by monocytes, because DNA samples were prepared from PBMCs in this study cohort. Profiles of mutated genes in PRCA appear to be different from those of aplastic anemia that were previously reported by other groups. It is yet to be known whether this could result from the different nature of both diseases, or the difference in the experimental protocols. Our findings strongly encourage conducting a prospective study to confirm our observation and clarify the diagnostic and predictive values of somatic mutations of myeloid malignancy-associated genes in acquired PRCA. This project is ongoing in collaboration with the prospective cohort study PRCA2016 being conducted in Japan.
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Nakao:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Matsuda:GlaxoSmithKline K.K.: Honoraria; Novartis Pharma K. K.: Honoraria; Chugai Pharmaceutical Co, Ltd.: Honoraria; Kyowa Hakko Kirin Co, Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Sanofi K.K.: Honoraria; Beckman Coulter K.K.: Honoraria. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau; Bristol-Myesr Squibb: Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Chugai: Research Funding; Astellas: Research Funding; Sumitomo Dainippon: Research Funding; Novartis: Research Funding; Toyama Chemical: Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Efficacy of Amikacin Combinations for Nocardiosis Kanemitsu, Keiji; Kunishima, Hiroyuki; Saga, Tomoo ...
The Tohoku Journal of Experimental Medicine,
11/2003, Volume:
201, Issue:
3
Journal Article
Peer reviewed
Open access
We isolated five bacterial strains from patients diagnosed as having nocardiosis. Bacterial species were identified based on the similarities in the nucleotide sequences of 16S ribosomal RNAs. Three ...of the five strains were identified as Nocardia asteroids, but unexpectedly other two were Streptomyces hygroscopicus and Rothia dentocariosa. The latter two species are not members of the family Nocardiaceae. We investigated the susceptibilities of these five strains to the following nine antimicrobial agents: trimethoprim/sulfamethoxazole (TMP/SMX), minocycline (MINO), erythromycin (EM), amikacin (AMK), cefotaxime (CTX), faropenem (FRPM), imipenem (IPM), ciprofloxacin (CPFX), and sparfloxacin (SPFX). The minimum inhibitory concentration (MIC) ranges (mg/ml) were as follows: TMP-SMX, 4->32; MINO, 0.125-8; EM, ≤0.016->32; AMK, 1-2; CTX, 0.063->32; FRPM, 0.063-16; IPM, 0.125-2; CPFX, 4-32; and SPFX, 0.5-16. Moreover, the synergistic effects of AMK in combination with each of TMP-SMX, MINO, EM, CTX, IPM, and SPFX were investigated by checkerboard synergy testing. No antagonism was recognized for the three N. asteroides strains. Synergistic and additive effects were observed for the combinations of AMK with CTX, IPM, or SPFX.
Candidemia remains a major cause of morbidity and mortality, especially in immunocompromised patients. A strategy of reducing virulence and virulence factors of Candida spp. is an attractive approach ...for the treatment of serious infections caused by these yeasts. Recently, farnesol has been reported to be a quorum-sensing autoinducer, as well as a virulence factor of C. albicans. In the present study, we examined the effects of pravastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor on the in vitro production of farnesol. In addition, the synergistic effects of pravastatin with fluconazole (FLC) were examined in a mouse model of systemic infections. In vitro experiments demonstrated that pravastatin had synergistic activity with FLC as judged by fractional inhibitory concentration index (FICI) and suppression of farnesol production at sub-minimum inhibitory concentrations. Furthermore, significant improvement of survival in systemic infection models was shown with pravastatin supplementation. The survival benefits of pravastatin were correlated with reductions of fungal burden. These data suggest the potential of pravastatin as a supportive therapy against C. albicans infections. Synergistic antifungal activity and suppression of HMG-CoA reductase-associated Candida virulence factors, including farnesol, may explain, at least in part, the in vivo efficacy of pravastatin.
We investigate the evolving molecular epidemiology of metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan ...from 2004 to 2006. MBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. bla.sub.IMP-1 group was the most predominant (38 isolates, 80%), followed by 3 bla.sub.IMP-7, 2 bla.sub.IMP-11 group, and 1 bla.sub.VIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole bla.sub.IMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with bla.sub.IMP-11 group or ST235 with bla.sub.IMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp. Our data give an important "snapshot" of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.
Full text
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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