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Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important in infection control, since it transmits plasmids carrying resistance genes. Here, we evaluated the rapid detection of CPE using the fully automated antimicrobial susceptability testing system "RAISUS S4". Sixty-two CPE strains including carbapenem-resistant Enterobacterales and 100 carbapenemase-non-producing Enterobacterales strains were used. RAISUS S4 was performed using both 18 hr and rapid methods. The sensitivity of CPE detection and decision time were evaluated using Meropenem results. The results showed that the sensitivity for CPE detection was 100% for both methods, with specificity of 97% for the 18 hr method and 95% for the rapid method. The mean CPE detection time for the 18 hr method was 7.2 hrs and 8.8 hrs for the rapid method. The mean MIC determination time of the 18 hr method for all 162 strains was 17.2 hrs and 8.8 hrs for the rapid method. In addition, we analyzed the absorbance values of the 18 hr method. Checking the growth curve at a drug concentration of 0.125 µg/ml and determining it to be positive when its absorbance reached 0.8 Abs, CPE could be detected in an average of 5.8 hrs with in sensitivity of 100% and specificity of 92%. The 18 hr method of RAISUS S4 allowed rapid detection of CPE, and the rapid method allowed earlier MIC determination, including sensitive isolates. These results suggest that RAISUS S4 can detect CPE rapidly without missing CPE by routine test even in Japan where the frequency of CPE isolation is low.

The emergence and dissemination of drug-resistant Gram-negative bacilli have been recognized as a serious health concern in worldwide. The isolation rates of Extended-Spectrum β-lactamases (ESBL) and AmpC β-lactamases (AmpC) producing gram negative rods are increasing in our hospital. In the present study, we evaluate the availability of the antimicrobial resistance testing by the direct disc methods using AmpC/ESBL differential discs. One hundred and ten strains of Enterobacterales were isolated during the observation period, of which 19 strains (17%) were ESBL-positive and 6 strains (5%) were AmpC-positive. The positive and negative coincidence rate between direct disc methods and standard disc methods were 100%. We conclude that the direct disc method is a useful and rapid detection method for ESBL and AmpC from blood culture samples.

Antimicrobial resistance in bacterial infections is a major concern for clinical settings. In recent years, the number of Extended-spectrum β-lactamase producing (ESBL)- and fluoroquinolones (FQ)-resistant Escherichia coli has been increasing in Japan, especially against third-generation cephalosporins and FQs, which are frequently used in medical practice. On the other hand, antimicrobial agents such as tazobactam-piperacillin, colistin, and tigecycline, which are not general-purpose agents but last-line drugs for multidrug-resistant bacteria, are also important. Enterobacteriaceae that are resistant to these antimicrobials have been reported, although the isolation rate of resistant bacteria is lower than that of frequent used antimicrobial resistance. The author has been studying antimicrobial drug resistance and multidrug resistance of bacteria isolated from clinical settings. In particular, bacteriological analysis of antimicrobial resistance, which is important for treatment, has been conducted mainly on E. coli isolated from clinical specimens at medical facilities in Sapporo City. In this article, the author describes the findings obtained so far.

There is a report that an infection by medicine resistant bacteria will be the number one cause of death in 2050 according to the recommendation of WHO, and the CPE (carbapenem-producing Enterobacteriaceae ) infection is regarded as a problem in particular. When detecting CPE, it is important how to detect stealth type CPE sensitive to carbapenem series medicines. So we used the 2 types of screening culture medium, "KBM" CRE-JU culture medium ‹KOJINBAIO› (CRE-JU culture medium) and the FRPM culture medium, and tried to detect drug-resistant gram-negative bacilli such as CPE, stealth type CPE, ESBL-producing bacteria, and excess AmpC-producing bacteria (AmpC-producing bacteria), etc. in combination of this culture mediums. As a result, CRE-JU culture medium showed a difference in the growth of CPE depending on the amount of inoculated bacteria while β-lactamase non-producing strain and other strains except for high concentration ESBL-producing bacteria and AmpC-producing bacteria were un-growing. Most of the CRE, stealth type CPE, ESBL-producing bacteria and AmpC-producing bacteria grew in the FRPM culture medium while most of the β-lactamase non-producing strains with a MIC value of meropenem (MEPM) of 2 µg/mL or less were un-growing. From these results, it was suggested that when a strain grown on CRE-JU and FRPM culture mediums, it could be distinguished as CPE, and when strains grown on FRPM culture medium which were un-grown on CRE-JU culture medium, it could be distinguished as drug-resistant bacteria such as stealth type CPE, ESBL-producing bacteria, and AmpC-producing bacteria. When strains not grown on CRE-JU and FRPM culture mediums, it could be distinguished as sensitive.

A 33-year-old male was admitted to our hospital due to bilateral optic neuritis (ON) and transverse myelitis (TM), which occurred almost simultaneously. Spinal MRI showed the longitudinally extensive TM, located from C2 to conus. Serum anti-aquaporin 4 antibody was negative. He was tentatively diagnosed as seronegative neuromyelitis optica spectrum disorders (NMOSD). During the clinical course, serum anti-myelin oligodendrocyte glycoprotein (MOG) antibody was detected, and finally he was diagnosed as anti-MOG antibody positive neurologic disease (MOG-ND). Our case highlighted that early detection of MOG antibody should be considered in male cases with clinical manifestation of classical Devic's disease, such as simultaneous disease onset of bilateral ON or ON + TM.

There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Metallo-β-lactamases (MBLs) are di-Zn(II) metalloenzymes that efficiently hydrolyze most β-lactam antibiotics used in clinical settings. Bacteria producing MBLs have been isolated from clinical settings and from natural environments such as rivers and soils, and are now recognized as a new potential threat to human health. No effective inhibitors are available for clinical use, making the treatment of infectious diseases caused by bacteria producing MBLs more difficult. IMP-1 is encoded on a plasmid which can be horizontally transferred between bacterial strains. Our studies on MBLs, and especially on IMP-1, focus on understanding the role of Zn(II) ion(s) in the hydrolysis of β-lactam antibiotics and on the detailed structure of the IMP-1 active site in order to develop efficient inhibitors. We investigated the role of the two Zn(II) ions in IMP-1 by kinetic, spectroscopic and thermodynamic analyses. The results revealed that the first Zn(II) ion is necessary for the hydrolysis of β-lactam antibiotics while the second Zn(II) ion enhances enzyme activity and structural stability, thus helping the enzyme achieve maximum activity. The detailed structures of the IMP-1 active site were examined by X-ray crystallography. Thiol compounds for irreversibly inhibiting IMP-1 were developed and the binding mode of these inhibitors was investigated in detail. These findings will aid the design of inhibitors that target MBLs.

Carbapenem is the last resort for infections due to Gram-negative bacteria. However, carbapenem-resistant Enterobacteriaceae such as KPC-producing bugs were reported in the United States from early this decade and now worldwide. According to the nationwide surveillance study in Japan performed between September and December, 2010, two bla(KPC)-harboring Klebsiella pneumoniae were identified among 153 multi-drug-resistant Enterobacteriaceae. Moreover, 72 out of 153 multi-drug-resistant Enterobacteriaceae harbored bla(IMP-1). The results of this surveillance study demonstrated that the most epidemic carbapenemase in Japan is not bla(KPC) but bla(LMP-1). Although the bla(KPC)-harboring strain is very rare in Japan, detection of KPC-producing Gram-negative bacteria may be difficult based on the CLSI recommended method. Development of effective screening methods is needed in the Japanese healthcare environment.

We analyzed the incidence of beta-lactamase non-producing ampicillin-resistance (BLNAR) Haemophilus influenzae in Showa University Hospital from June 2004 to March 2005. The ratio of BLNAR in total H. influenzae isolates was 44.5%. BLNAR accounted for 89.6% of the ampicillin non-susceptible strains displaying a minimal inhibitory concentration(MIC) of ampicillin of more than 2 microg/ml, which is the break point advocated by the Clinical and Laboratory Standards Institute. Of the total BLNARs, 71.4% were isolated from children of six and under. We analyzed mutations of the SSN and KTG motif of penicillin binding protein 3 encoded by the ftsI gene, since mutations in these regions are considered to be responsible for the drug-resistance of BLNAR. Mutations in SSN and KTG motifs were identified in all BLNAR isolates. We also detected mutations in the ampicillin susceptible strains displaying an ampicillin MIC of 0.5 or microg/ml. When a noticeable MIC increase of ABPC or other beta-lactams in the routine clinical laboratory practice, gene analysis of ftsI gene of the isolates displaying increased MIC is required to measure the spreading of BLNAR.

Haemophilus influenzae type b (Hib) is the most frequent pathogen of bacterial meningitis in Japanese children. The prevalence of beta-lactamase-negative ampicillin-resistant (BLNAR) Hib strain has been increasing in recent years. Furthermore, antibiotic activities of cefotaxime (CTX) and ceftriaxone (CTRX) have decreased against some of those BLNAR strains. We report a case of one-year-old boy who suffered from meningitis caused by BLNAR Hib. The MICs of CTX and CTRX for the strain isolated from cerebrospinal fluid was 1.0 and 0.5 microg/ml, respectively. The patient was administered high-dose CTRX (150 mg/kg/day) and recovered completely without any sequela. The high-dose CTRX administration may be a considerable choice of the treatment of BLNAR meningitis.

We experienced a case in the infant of the bacterial meningitis due to beta-lactamase negative ampicillin-resistant Haemophilus influenzae (BLNAR) which has increased in recent years. In the present condition, the sensitivity of the bacteria to the antibacterial-drug used as the initial treatment for the bacterial meningitis is sometimes inadequate. If it takes into consideration that BLNAR participates in H. influenzae meningitis, it will be one of the choice to use meropenem with other antibacterial-drug concomitantly.

The following results were obtained from the comparison of cefoperazone (CPZ) with cefazolin (CEZ), cephalothin (CET) and cefoxitin (CFX) in respect to their antibacterial action against anaerobic bacteria: 1) CPZ showed an antibacterial spectrum which was comparable to those of the known antibiotics, CEZ, CET and CFX. CPZ was particularly effective against anaerobic Gram-positive bacteria. 2) CPZ showed a distribution of susceptibility against clinically isolated strains which was comparable to those of CEZ, CET and CFX, CPZ was slightly inferior to CFX against B. fragilis and B. thetaiotaomicron, but was somewhat superior to any of CEZ, CET and CFX against anaerobic Gram-positive rods and B. distasonis. 3) The susceptibility of B. fragilis and F. necrophorum to CPZ decreased with an increase in the amount of inoculation, while C. perfringens and P. asaccharolyticus did not show any difference in susceptibility with the amount of inoculation. 4) The values of MIC and MBC of CPZ showed good coincidence with each other, thereby testifying its superior bactericidal action. 5) Natural resistant mutants were not obtained. 6) According to the examination of resistance in a test tube, P. variabilis did not show any appreciable increase in resistance, but F. varium increased its resistance rapidly. 7) CPZ was inferior only to CFX in stability against the beta-lactamase of B. fragilis. All of CEZ, CET and CMD lacked stability against the beta-lactamase of B. fragilis. 8) CPZ was found effective for the treatment of experimental subcutaneous abscess in mice caused by F. necrophorum.