Kenji Marumo

Purification and Characterization of Metallo-β-Lactamase from Serratia marcescens

Carbapenem‐hydrolyzing β‐lactamase from Serratia marcescens FHSM4055 was purified 926‐fold by means of carboxylmethyl Sephadex C‐50, Sephacryl S‐200, and Mono S column chromatography. The molecular weight was 30,000 by SDS‐PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p‐chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo‐β‐lactamase and that the SH‐group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (kcat/Km) showed that the enzyme hydrolyzed various β‐lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino‐ and imino‐groups, ceftazidime with a carboxypropyloxyimino‐group, and cefclidin with a carbamoylquinuclidine‐group were poor substrates among the β‐lactam antibiotics other than the monobactams tested. The plots of the turnover number (kcat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK1, 5.9; pK2, 9.0; pK3, 10.8), whereas those of kcat/Km gave a bell‐shaped curve (pK1, 5.8; pK2, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N‐terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71‐78), the above enzymatic characteristics seem to coincide.

Genetic Characteristics of CTX-M-Type Extended-Spectrum-β-Lactamase (ESBL)-Producing Enterobacteriaceae Involved in Mastitis Cases on Japanese Dairy Farms, 2007 to 2011

ABSTRACT Sixty-five CTX-M-2/15/14 extended-spectrum-β-lactamase-producing Enterobacteriaceae were isolated from 258,888 mastitic milk samples from Japanese dairy farms between 2007 and 2011. CTX-M-2-producing Klebsiella pneumoniae and CTX-M-15-producing Escherichia coli were the predominant strains isolated. There was no predominant clonal type, and clonal diversity was found even in strains isolated from a single farm.

Herd prevalence of Enterobacteriaceae producing CTX-M-type and CMY-2 β-lactamases among Japanese dairy farms.

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak.

Aims:  To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds.

Isolation of novel mycobacteria contaminating an aquarium fish tank in a Japanese university hospital

Aims:  To better understand nontuberculous mycobacteria (NTM) contamination in a hospital setting, six freshwater fish gut homogenates and water in an aquarium fish tank placed on the reception counter of a nursing station were cultured for mycobacteria.