First VanD-Type vancomycin-resistant Enterococcus raffinosus isolate.
Explore the source record for details and available documents.
Aged↗
Biomedical subjects
Naohiro Shibata
Publications and source records attributed to Naohiro Shibata.
[Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla(IMP-1) and bla(VIM-2) genes].
Loop-mediated isothermal amplification (LAMP) amplifies a target gene with high specificity and rapidity under isothermal conditions. LAMP assays were developed for the rapid detection of metallo-beta-lactamase (MBL) genes such as bla(IMP-1)) and bla(VIM-2). We initially designed specific primers to detect MBL genes for LAMP assays and evaluated the specificity and sensitivity of these assays. LAMP assays amplified MBL genes under a constant temperature of 63 degrees C within 1 hour, and were compared to PCR in MBL-producing strains. The results of MBL genes typing by LAMP assays agree completely with PCR results. The lower detection limits of bla(IMP-1)- and bla(VIM-2)-LAMP assays using real-time turbidimeters were 30cfu/test and 3cfu/test. After amplification, products were directly observed by the naked eye with a fluorescent detection reagent. In conclusion, LAMP assays are convenient, rapid, and fully feasible for detecting MBL genes in ordinary clinical microbiology laboratories without special apparatus.
Gene Amplification↗
[One case with Erysipelothrix rhusiopathiae endocarditis].
We report case of a 67-year-old alcoholic fisherman who developed infective endocarditis caused by Erysipelothrix rhusiopathiae. The initial manifestations were fever and back pain of approximately three months' duration. Auscultation of the heart revealed a loud systolic murmur at the apex and a diastolic murmur over the aortic valve area. Echocardiographic studies showed vegetations on both the aortic valve and mitral valve. Blood cultures grew Erysipelothrix rhusiopathiae, and it was sensitive to aminobenzyl penicillin. No other bacteria grew aut. A diagnosis of infective endocarditis caused by the Erysipelothnx rhusiopatniae was made, and the patient was treated with aminobenzyl penicillin 12g/day for 6 weeks. His clinical course was complicated by heart failure, multiple cerebral embolism, and renal infarctions. However, he recovered without valve replacement. Although the exact route of infection remains unknown, erosions of the skin, of his palms at the time of tho initial examination appeared to be one possible source of the systemic infection in this case. The serotype of the bacteria was Ib. To our knowledge this is the first case of serotyping of bacterium that caused endocarditis in humans.
Aged↗
Mode of transposition and expression of 16S rRNA methyltransferase gene rmtC accompanied by ISEcp1.
A newly identified 16S rRNA methyltransferase gene, rmtC, was accompanied by an ISEcp1 element at its 5' end. This ISEcp1 element, which contained a transposase gene, tnpA, provided a promoter activity for expression of the adjacent rmtC; and this structure enabled the rmtC gene to be transposed onto another plasmid in Escherichia coli.
Base Sequence↗
Novel plasmid-mediated 16S rRNA methylase, RmtC, found in a proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides.
Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DH5alpha was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (< or = 29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (< or = 28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed methyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing tnpA. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future.
Amino Acid Sequence↗
Horizontal transfer of blaCMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19.
Nine Escherichia coli and 5 Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital between 1995 and 1997. All nine E. coli isolates and one K. pneumoniae isolate carried bla(CMY-9), while the other four K. pneumoniae isolates harbored a variant of bla(CMY-9), namely, bla(CMY-19). The pulsed-field gel electrophoresis patterns of the nine CMY-9-producing E. coli isolates were almost identical, suggesting their clonal relatedness, while those of the five K. pneumoniae isolates were divergent. Plasmid profiles, Southern hybridization, and conjugation assays revealed that the genes for the CMY-9 and the CMY-19 beta-lactamases were located on very similar conjugative plasmids in E. coli and K. pneumoniae. The genetic environment of bla(CMY-19) was identical to that of bla(CMY-9). A single amino acid substitution, I292S, adjacent to the H-10 helix region was observed between CMY-9 and CMY-19. This substitution was suggested to be responsible for the expansion of the hydrolyzing activity against several broad-spectrum cephalosporins, and this finding was consistent with the kinetic parameters determined with purified enzymes. These findings suggest that the bla(CMY-19) genes found in the four K. pneumoniae isolates might have originated from bla(CMY-9) gene following a point mutation and dispersed among genetically different K. pneumoniae isolates via a large transferable plasmid.
Amino Acid Sequence↗
PCR classification of CTX-M-type beta-lactamase genes identified in clinically isolated gram-negative bacilli in Japan.
Of 1,456 strains isolated from 2001 to 2003 demonstrating resistance to either oxyimino-cephalosporin, 317 strains, isolated in 57 of 132 clinical facilities, were found to harbor bla(CTX-M) genes by PCR. Fifty-seven, 161, and 99 strains harbored bla(CTX-M) genes belonging to the bla(CTX-M-1), bla(CTX-M-2), and bla(CTX-M-9) clusters, respectively.
Escherichia coli Proteins↗
A nosocomial outbreak due to novel CTX-M-2-producing strains of Citrobacter koseri in a hematological ward.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
Practical methods using boronic acid compounds for identification of class C beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli.
Detection of the resistance mediated by class C beta-lactamases remains a challenging issue, considering that transferable plasmid-mediated class C beta-lactamases are of worldwide concern. Methods for the identification of strains that produce extended-spectrum beta-lactamases (ESBLs) or metallo-beta-lactamases (MBLs) have been developed and applied for routine use in clinical microbiology laboratories, but no practical methods for identification of plasmid-mediated class C producers have been established to date. We therefore developed three simple methods for clinical microbiology laboratories that allow identification of plasmid-mediated class C beta-lactamase-producing bacteria using a boronic acid derivative, 3-aminophenylboronic acid (APB), one of the specific inhibitors of class C beta-lactamases. Detection by the disk potentiation test was based on the enlargement of the growth-inhibitory zone diameter (by greater than or equal to 5 mm) around a Kirby-Bauer disk containing a ceftazidime (CAZ) or a cefotaxime (CTX) disk in combination with APB. In a double-disk synergy test, the discernible expansion of the growth-inhibitory zone around the CAZ or the CTX disk toward a disk containing APB was indicative of class C beta-lactamase production. A greater than or equal to eightfold decrease in the MIC of CAZ or CTX in the presence of APB was the criterion for detection in the microdilution test. By using these methods, Escherichia coli and Klebsiella pneumoniae isolates producing plasmid-mediated class C beta-lactamases, ACT-1, CMY-2, CMY-9, FOX-5, LAT-1, and MOX-1, were successfully distinguished from those producing other classes of beta-lactamases, such as ESBLs and MBLs. These methods will provide useful information needed for targeted antimicrobial therapy and better infection control.
Anti-Bacterial Agents↗
Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides.
Serratia marcescens S-95, which displayed an unusually high degree of resistance to aminoglycosides, including kanamycins and gentamicins, was isolated in 2002 from a patient in Japan. The resistance was mediated by a large plasmid which was nonconjugative but transferable to an Escherichia coli recipient by transformation. The gene responsible for the aminoglycoside resistance was cloned and sequenced. The deduced amino acid sequence of the resistance gene shared 82% identity with RmtA, which was recently identified as 16S rRNA methylase conferring high-level aminoglycoside resistance in Pseudomonas aeruginosa. Histidine-tagged recombinant protein showed methylation activity against E. coli 16S rRNA. The novel aminoglycoside resistance gene was therefore designated rmtB. The genetic environment of rmtB was further investigated. The sequence immediately upstream of rmtB contained the right end of transposon Tn3, including bla(TEM), while an open reading frame possibly encoding a transposase was identified downstream of the gene. This is the first report describing 16S rRNA methylase production in S. marcescens. The aminoglycoside resistance mechanism mediated by production of 16S rRNA methylase and subsequent ribosomal protection used to be confined to aminoglycoside-producing actinomycetes. However, it is now identified among pathogenic bacteria, including Enterobacteriaceae and P. aeruginosa in Japan. This is a cause for concern since other treatment options are often limited in patients requiring highly potent aminoglycosides such as amikacin and tobramycin.
Aged↗
Nosocomial spread of ceftazidime-resistant Klebsiella pneumoniae strains producing a novel class a beta-lactamase, GES-3, in a neonatal intensive care unit in Japan.
Klebsiella pneumoniae strain KG525, which showed high-level resistance to broad-spectrum cephalosporins, was isolated from the neonatal intensive care unit (NICU) of a Japanese hospital in March 2002. The ceftazidime resistance of strain KG525 was transferable to Escherichia coli CSH-2 by conjugation. Cloning and sequence analysis revealed that production of a novel extended-spectrum class A beta-lactamase (pI 7.0), designated GES-3, which had two amino acid substitutions of M62T and E104K on the basis of the sequence of GES-1, was responsible for resistance in strain KG525 and its transconjugant. The bla(GES-3) gene was located as the first gene cassette in a class 1 integron that also contained an aacA1-orfG fused gene cassette and one unique cassette that has not been described in other class 1 integrons and ended with a truncated 3' conserved segment by insertion of IS26. Another five ceftazidime-resistant K. pneumoniae strains, strains KG914, KG1116, KG545, KG502, and KG827, which were isolated from different neonates during a 1-year period in the same NICU where strain KG525 had been isolated, were also positive for GES-type beta-lactamase genes by PCR. Pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR analyses displayed genetic relatedness among the six K. pneumoniae strains. Southern hybridization analysis with a GES-type beta-lactamase gene-specific probe showed that the locations of bla(GES) were multiple and diverse among the six strains. These findings suggest that within the NICU setting genetically related K. pneumoniae strains carrying the bla(GES) gene were ambushed with genetic rearrangements that caused the multiplication and translocation of the bla(GES) gene.
Amino Acid Sequence↗
Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates.
Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997. These strains harbor the newly identified 16S rRNA methylase gene (rmtA). When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed. The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared. In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na(+)/H(+) antiporter gene-like sequence (sequence 2) was identified downstream of rmtA. This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp kappagamma elements. Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus. In strain AR-11, the rmtA gene and sequence 2 were found, but the 5' end of the orfA gene was truncated and replaced with IS6100. An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11. The G+C content of the rmtA gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future.
Anti-Bacterial Agents↗
Spread of novel aminoglycoside resistance gene aac(6')-Iad among Acinetobacter clinical isolates in Japan.
A novel aminoglycoside resistance gene, aac(6')-Iad, encoding aminoglycoside 6'-N-acetyltransferase, was identified in Acinetobacter genospecies 3 strain A-51. The gene encoded a 144-amino-acid protein, which shared modest identity (up to 36.7%) with some of the aminoglycoside 6'-N-acetyltransferases. The results of high-pressure liquid chromatography assays confirmed that the protein is a functional aminoglycoside 6'-N-acetyltransferase. The enzyme conferred resistance to amikacin, tobramycin, sisomicin, and isepamicin but not to gentamicin. The prevalence of this gene among Acinetobacter clinical isolates in Japan was then investigated. Of 264 Acinetobacter sp. strains isolated from geographically diverse areas in Japan in 2002, 16 were not susceptible to amikacin, and aac(6')-Iad was detected in 7. Five of the producers of aminoglycoside 6'-N-acetyltransferase type Iad were identified as Acinetobacter baumannii, and two were identified as Acinetobacter genospecies 3. These results suggest that aac(6')-Iad plays a substantial role in amikacin resistance among Acinetobacter spp. in Japan.
Acetyltransferases↗
Inhibitor-sensitive AmpC beta-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins.
Escherichia coli HKY28, a ceftazidime-resistant strain isolated from a urine specimen in Japan, produced an inhibitor-sensitive AmpC beta-lactamase variant. The deduced amino acid sequence of the enzyme contained a number of substitutions and a tripeptide deletion (Gly286-Ser287-Asp288) compared with the sequence of native AmpC of E. coli. When the deletion was reverted by a 9-base insertion at the relevant site of ampC in the clone, the typical inhibitor-resistant phenotype of AmpC was restored, while at the same time the levels of resistance to ceftazidime, cefpirome, and cefepime were reduced eightfold or more. Molecular modeling studies indicated that a structural change took place in the H-10 helix as a result of the deletion, and this change caused an alteration of the substrate binding site, leading to a unique phenotype analogous to that of inhibitor-sensitive class A extended-spectrum beta-lactamases. The degree of inhibition was greater with sulbactam and tazobactam than with clavulanic acid. To our knowledge, this is the first report to have characterized an E. coli ampC that encodes chromosomal AmpC beta-lactamase sensitive to the available beta-lactamase inhibitors.
Amino Acid Sequence↗
Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A beta-lactamase, GES-4, possessing a single G170S substitution in the omega-loop.
The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.
Amino Acid Sequence↗
Metallo-beta-lactamase-producing gram-negative bacilli: laboratory-based surveillance in cooperation with 13 clinical laboratories in the Kinki region of Japan.
A total of 19,753 strains of gram-negative rods collected during two 6-month periods (October 2000 to March 2001 and November 2001 to April 2002) from 13 clinical laboratories in the Kinki region of Japan were investigated for the production of metallo-beta-lactamases (MBLs). MBLs were detected in 96 (0.5%) of the 19,753 isolates by the broth microdilution method, the 2-mercaptopropionic acid inhibition test, and PCR and DNA sequencing analyses. MBL-positive isolates were detected in 9 of 13 laboratories, with the rate of detection ranging between 0 and 2.6% for each laboratory. Forty-four of 1,429 (3.1%) Serratia marcescens, 22 of 6,198 (0.4%) Pseudomonas aeruginosa, 21 of 1,108 (1.9%) Acinetobacter spp., 4 of 544 (0.7%) Citrobacter freundii, 3 of 127 (2.4%) Providencia rettgeri, 1 of 434 (0.2%) Morganella morganii, and 1 of 1,483 (0.1%) Enterobacter cloacae isolates were positive for MBLs. Of these 96 MBL-positive strains, 87 (90.6%), 7 (7.3%), and 2 (2.1%) isolates carried the genes for IMP-1-group MBLs, IMP-2-group MBLs, and VIM-2-group MBLs, respectively. The class 1 integrase gene, intI1, was detected in all MBL-positive strains, and the aac (6')-Ib gene was detected in 37 (38.5%) isolates. Strains with identical PCR fingerprint profiles in a random amplified polymorphic DNA pattern analysis were isolated successively from five separate hospitals, suggesting the nosocomial spread of the organism in each hospital. In conclusion, many species of MBL-positive gram-negative rods are distributed widely in different hospitals in the Kinki region of Japan. The present findings should be considered during the development of policies and strategies to prevent the emergence and further spread of MBL-producing bacteria.
Anti-Bacterial Agents↗
Nosocomial transmission of CTX-M-2 beta-lactamase-producing Acinetobacter baumannii in a neurosurgery ward.
Three strains of cefotaxime (CTX)-resistant Acinetobacter baumannii, FM0209680, FM0300106, and FM0301433, were isolated from transtracheal aspirate cultures of three patients with probable nosocomial infections in a neurosurgery ward in Japan. The CTX MICs for these isolates were greater than 128 microg/ml but were drastically reduced in the presence of 4 microg of clavulanic acid per ml. These strains were also resistant to ceftriaxone, cefpodoxime, and aztreonam but were susceptible to ceftazidime and imipenem. The profile of resistance to various broad-spectrum beta-lactams was transferred by conjugation. Strain FM0209680 was not eradicated from case patient 1 by administration of imipenem, ceftazidime, and levofloxacin, even after a 6-month hospitalization period. Strains FM0300106 and FM0301433 were isolated from case patients 2 and 3 during the sixth week following admission, respectively, and then each patient was colonized for 3 weeks. Eradication of FM0300106 was successfully obtained from case patient 2 by imipenem treatment, while administration of imipenem was continued to prevent pneumonia. Prophylactic antimicrobial therapy was discontinued in case patient 3 because of the lack of pneumonic symptoms, and FM0301433 disappeared after the discontinuation of antimicrobial chemotherapy. All three strains carried the bla(CTX-M-2) gene, and the appearance of colonies in the growth-inhibitory zones around disks of CTX and aztreonam in double-disk synergy tests suggested inducible beta-lactamase production in these A. baumannii strains. The ribotyping investigation suggested that all these strains belong to the same clonal lineage. The plasmids harbored by A. baumannii had the same restriction profile as those harbored by Proteus mirabilis strains previously isolated in a urology ward of the Funabashi Medical Center.
Acinetobacter Infections↗
Comparative study of the inhibition of metallo-beta-lactamases (IMP-1 and VIM-2) by thiol compounds that contain a hydrophobic group.
For the purpose of screening of inhibitors that are effective for wide range of metallo-beta-lactamases, the inhibitory effect of two series of compounds, 2-omega-phenylalkyl-3-mercaptopropionic acid (PhenylCnSH (n=1-4)) and N-[(7-chloro-quinolin-4-ylamino)-alkyl]-3-mercapto-propionamide (QuinolineCnSH (n=2-6)), where n denotes the alkyl chain length, on metallo-beta-lactamases IMP-1 and VIM-2 was examined. These inhibitors contain a thiol group and a hydrophobic group linked by variable-length methylene chain. PhenylCnSH (n=1-4) was found to be a potent inhibitor of both IMP-1 and VIM-2. PhenylC4SH was the potent inhibitor of both IMP-1 (IC(50)=1.2 microM) and VIM-2 (IC(50)=1.1 microM) among this study. When the number of methylene units was varied, QuinolineC4SH showed the maximum inhibitory activity against IMP-1 and VIM-2 (IC(50)=2.5 microM and IC(50)=2.4 microM). The relationship between the inhibitory effect of the alkyl chain length was different for both series of inhibitors, suggesting that IMP-1 has a tighter binding site than VIM-2. QuinolineCnSH did not serve as a fluorescence reagent for metallo-beta-lactamases.
Dose-Response Relationship, Drug↗