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Yoko Takayama, 1, 2 Tsuyoshi Sekizuka, 3 Hidehito Matsui, 4 Yuzuru Adachi, 4 Ryotaro Eda, 5 Shin Nihonyanagi, 2 Tatsuhiko Wada, 2 Mari Matsui, 6 Satowa Suzuki, 6 Masashi Takaso, 7 Hidero Kitasato, 5 Makoto Kuroda, 3 Hideaki Hanaki 4 1Department of Infection Control and Infectious Diseases, Research and Development Center for New Medical Frontiers, Kitasato University School of Medicine, Kanagawa, Japan; 2Department of Infection Control and Prevention, Kitasato University Hospital, Kanagawa, Japan; 3Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan; 4Infection Control Research Center, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan; 5Department of Microbiology, School of Allied Health Sciences, Kitasato University, Kanagawa, Japan; 6Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Tokyo, Japan; 7Department of Orthopaedic Surgery, Kitasato University School of Medicine, Kanagawa, JapanCorrespondence: Yoko Takayama 1-15-1 Kitasato, Minami-Ku, Sagamihara, Kanagawa 252-0374, JapanTel/Fax +81-42-778-9119Email yoko@med.kitasato-u.ac.jpPurpose: New Delhi metallo-β-lactamase 5 (NDM-5) shows stronger resistance to carbapenems and broad-spectrum cephalosporins than NDM-1 because NDM-5 differs from NDM-1 by two amino acid substitutions. In this study, our aim was to characterize a NDM-5-producing Escherichia coli isolate KY1497 from a patient with urinary tract infection in Japan, who had no recent history of overseas travel.Patients and Methods: NDM-5-producing E. coli isolate KY1497 was detected in the urine sample of a patient hospitalized in a tertiary hospital in Japan. The complete genome sequence of isolate KY1497 was determined by short- and long-read sequencing with hybrid assembly, followed by multilocus sequence typing (MLST), core-genome phylogeny analysis, plasmid analysis, and transconjugation experiments.Results: KY1497 was classified as ST405 by MLST, and core-genome phylogeny exhibited the closest lineage to the clinical isolates in Nepal (IOMTU605) and Canada (FDAARGOS_448). KY1497 harbors blaNDM-5 in the IncFII-IncFIB(pB171) replicon plasmid (pKY1497_1, 123,767 base pairs). Plasmid analysis suggested that the cognate plasmids of pKY1497_1 have a minor plasmid background, rather than the globally disseminated IncX3 plasmid carrying blaNDM-5. Transconjugation analysis revealed that pKY1497_1 is transmissible to the recipient E. coli J53 strain.Conclusion: We characterized a novel Inc replicon plasmid (IncFII-IncFIB[pB171]) carrying blaNDM-5 and its host E. coli strain. NDMs are associated with a high risk of infection worldwide because of their antibiotic resistance and untreatable and hard-to-treat infections. Other patients in the hospital showed negative results for carbapenem-resistant Enterobacteriaceae. As NDM-producing strains are only sporadically detected in Japan, attention should be provided to the community prevalence of NDM-producing E. coli strains to prevent nosocomial infections.Keywords: blaNDM-5, Escherichia coli, sequence type 405, IncFII, IncFIB

The form deviations of tooth flanks of spur and helical gears obtained by using a laser holographic interferometer are compared with the results of the conventional measuring method using a contacting stylus. The objective tooth flank which has a rough finish compared with optical parts is irradiated with a laser beam in a large incident angle to obtain the reflected ray from it. Image patterns of the interference fringes for tooth flanks with various types of form deviation are obtained, and they are transformed to the form deviations from the target tooth flank form. The algorithm of this transformation is shown: the brightness information of the image of the interference fringe on the CRT is converted to the amplitude of form deviation defined on the plane of action of the gear according to the phase difference of light beam by using the phase stepping method. A partial measuring procedure for helical gears is proposed which achieves the same level of accuracy as the conventional method using a stylus.

Abstract  For the purpose of a nationwide surveillance of the antimicrobial susceptibility of bacterial respiratory pathogens in patients in Japan, the Japanese Society of Chemotherapy conducted their second year survey, during the period from January to August, 2007. A total of 1178 strains were collected from clinical specimens obtained from adult patients with well-diagnosed respiratory tract infections. Susceptibility testing was evaluable for 1108 strains (226 Staphylococcus aureus, 257 Streptococcus pneumoniae, 6 Streptococcus pyogenes, 206 Haemophilus influenzae, 120 Moraxella catarrhalis, 122 Klebsiella pneumoniae, and 171 Pseudomonas aeruginosa). A total of 44 antibacterial agents, including 26 β-lactams (four penicillins, three penicillins in combination with β-lactamase inhibitors, four oral cephems, eight parenteral cephems, one monobactam, five carbapenems, and one penem), three aminoglycosides, four macrolides (including ketolide), one lincosamide, one tetracycline, two glycopeptides, six fluoroquinolones, and one oxazolidinone were used for the study. Analysis was conducted at the central reference laboratory according to the method recommended by the Clinical and Laboratory Standards Institute (CLSI). The incidence of methicillinresistant Staphylococcus aureus (MRSA) was high, at 59.7%, and the incidences of penicillin-intermediateresistant and -resistant Streptococcus pneumoniae (PISP and PRSP) were 30.4% and 5.1%, respectively. Among Haemophilus influenzae strains, 19.9% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately-resistant (BLNAI), 29.1% to be β-lactamasenon-producing ABPC-resistant (BLNAR), and 6.7% to be β-lactamase-producing ABPC-resistant (BLPAR) strains. Extended-spectrum β-lactamase-producing Klebsiella pneumoniae was not isolated. Two isolates (1.2%) of Pseudomonas aeruginosa were found to be metallo-β-lactamase-producing strains, including one (0.6%) suspected multidrug-resistant strain showing resistance to imipenem, amikacin, and ciprofloxacin. These data will be a useful reference for future periodic surveillance studies and for investigations to control resistant infections as well. Continued surveillance is required to prevent the further spread of these antimicrobial resistances. [ABSTRACT FROM AUTHOR]

More than 90% of methicillin-resistant Staphylococcus aureus (MRSA) isolates produce a penicillin-binding protein PBP2′ (or PBP2a) with low affinity for β-lactam antibiotics. PBP2′ is encoded by the mecA gene, a foreign gene integrated into the chromosome of methicillin-susceptible S. aureus (MSSA). DNA vaccination by injection of transgene-expressing plasmids has been demonstrated to elicit an immune response against transgene-encoded protein. We hypothesized that the application of DNA vaccination with the mecA sequence would elicit protective immunity against MRSA. This immunity was evoked by injection of a mecA-expressing plasmid into BALB/c mice. Anti-PBP2′ antibody was detected in the sera obtained from the DNA-vaccinated mice. These sera produced a five-fold increase in phagocytosis of MRSA compared with sera from mice treated with control plasmid. However, there was no difference in phagocytosis of MSSA among these groups. In addition, the in-vivo antibacterial effect of DNA vaccination was demonstrated in mice infected with MRSA. Eight days after iv inoculation of 108 cfu of MRSA into mice, the number of bacteria in the kidneys obtained from mice vaccinated with mecA-expressing plasmid (1.48 ± 0.27 × 105 cfu/mg kidney; n = 18) was significantly lower than that from mice vaccinated with negative control plasmid (3.59 ± 0.57 × 105 cfu/mg kidney; n = 17) (P < 0.02) or that from sham-treated mice (3.43 ± 0.66 × 105 cfu/mg kidney; n = 9) (P < 0.02). Interestingly, PBP2′ was found in both the bacterial membrane fraction and the supernatant, thus being accessible to serum antibodies. Together these observations indicate that PBP2′ or the mecA sequence may be eligible as a candidate molecule for vaccination against MRSA. [ABSTRACT FROM PUBLISHER]

The peptidoglycan compositions of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (MIC 8 mg/L) and hetero-VRSA strain Mu3 (MIC 3 mg/L) were compared in order to understand the mechanism of vancomycin resistance. As compared with Mu3, the cell wall of Mu50 had increased amounts of glutamine-non-amidated muropeptides and decreased cross-linking of peptidoglycan with a greatly decreased dimer/monomer ratio of muropeptides. In agreement with this observation, the peptidoglycan of Mu50 bound 1.4 times more vancomycin than that of Mu3. The increase in non-amidated muropeptides and the reduced cross-linking of the cell-wall peptidoglycan may contribute to the vancomycin resistance by increasing the consumption of vancomycin by the pre-existing cell wall of Mu50 and reducing the amount of vancomycin reaching the cytoplasmic membrane where the vital targets of the antibiotic are situated. [ABSTRACT FROM AUTHOR]

The peptidoglycan compositions of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (MIC 8 mg/L) and hetero-VRSA strain Mu3 (MIC 3 mg/L) were compared in order to understand the mechanism of vancomycin resistance. As compared with Mu3, the cell wall of Mu50 had increased amounts of glutamine-non-amidated muropeptides and decreased crosslinking of peptidoglycan with a greatly decreased dimer/monomer ratio of muropeptides. In agreement with this observation, the peptidoglycan of Mu50 bound 1.4 times more vancomycin than that of Mu3. The increase in non-amidated muropeptides and the reduced crosslinking of the cell-wall peptidoglycan may contribute to the vancomycin resistance by increasing the consumption of vancomycin by the preexisting cell wall of Mu50 and reducing the amount of vancomycin reaching the cytoplasmic membrane where the vital targets of the antibiotic are situated. [ABSTRACT FROM PUBLISHER]

We have previously reported methicillin-resistant Staphylococcus aureus clinical strains, Mu50 and Mu3, representing two categories of vancomycin resistance: Mu50 representing vancomycin-resistant S. aureus (VRSA) with MICs ≥8 mg/L, and Mu3 representing hetero-VRSA with MICs ≤4 mg/L using standard MIC determination methods. The mechanisms of vancomycin resistance in these strains were investigated. These strains did not carry the enterococcal vancomycin-resistance genes, vanA, vanB,or VanC1-3, as tested by PCR using specific primers. However, both strains produced three to five times the amount of penicillin-binding proteins (PBPs) 2 and 2′ when compared with vancomycin-susceptible S. aureus control strains with or without methicillin resistance; the amounts of PBP2 produced in Mu3 and Mu50 were comparable to those in the vancomycin-resistant S. aureus mutant strains selected in vitro. Incorporation of 14C-labelled N-acetyl-glucosamine into the cell was three to 20 times increased in Mu50 and Mu3, and release of the radioactive cell wall material was increased in Mu3 (and also in Mu50, though to a lesser extent), compared with control strains. The amounts of intracellular murein monomer precursor in these strains were three to eight times greater than those found in control strains. Transmission electron microscopy showed a doubling in the cell wall thickness in Mu50 compared with the control strains. Mu3 did not show obvious cell wall thickening. These data indicate that activated synthesis and an increased rate of cell wall turnover are common features of Mu3 and Mu50 and may be the prerequisite for the expression of vancomycin resistance in S. aureus. [ABSTRACT FROM PUBLISHER]

An imaging plate has been used as a useful detector of energetic electrons in laser electron acceleration and laser fusion studies. The absolute sensitivity of an imaging plate was calibrated at 1 GeV electron energy using the injector Linac of SPring-8. The sensitivity curve obtained up to 100 MeV in a previous study was extended successfully to GeV range. [ABSTRACT FROM AUTHOR]