Satoshi Ichiyama

Plasmid-mediated AmpC-type beta-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum beta-lactams, including moxalactam.

Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded beta-lactamase (MOX-1) which conferred resistance to broad spectrum beta-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10(-7). The structural gene of MOX-1 (blaMOX-1) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was > 512 micrograms/ml. The apparent molecular mass and pI of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg2+ and Cu2+ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine beta-lactamase inhibitors, inhibited the enzyme activity competitively (Kis = 5.60 and 0.35 microM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum beta-lactams. A hybridization study confirmed that blaMOX-1 is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX-1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type beta-lactamase that provides enteric bacteria resistance to broad-spectrum beta-lactams, including moxalactam. Images

The Isolation of Mycobacterium avium Complex from Soil, Water, and Dusts

Previously, it was difficult to isolate the Mycobacterium avium complex from soil, water, and dusts, because rapidly growing mycobacteria always overgrew slowly growing ones. We used Ogawa egg medium containing both ethambutol and ofloxacin, which inhibit the nonpathogenic slowly growing mycobacteria and most rapidly growing mycobacteria, respectively, as an aid to screen for pathogenic slowly growing mycobacteria; we could thereby isolate a number of the M. avium complex and M. scrofulaceum strains from soil, water, and dusts in this country.

Comparison of DNA fingerprinting by PFGE and PCR-RFLP of the coagulase gene to distinguish MRSA isolates

Staphylococcus aureus isolates were collected from epidemiologically unrelated clinical sources in Japan between 1991 and 1993. A total of 40 isolates, five each of eight coagulase types, were analysed by polymerase chain reaction (PCR) of the coagulase gene, PCR-restriction fragment length polymorphism (RFLP) after AluI digestion, and pulsed-field gel electrophoresis (PFGE) of chromosomal DNA after SmaI digestion. The efficiency of discrimination among the isolates increased in the order of PCR < PCR-RFLP < PFGE, yielding five, 13 and 31 different types, respectively. To assess the clinical use of these methods, 42 additional methicillin-resistant S. aureus (MRSA) isolates collected from 27 inpatients in a hospital were analysed. PFGE and PCR-RFLP were able to discriminate 11 and four types, respectively. PFGE analysis detected cross-infection between four postoperative patients in an intensive-care unit, and in six neonates in intensive care. We conclude that of the three methods tested, PFGE analysis currently allows the most effective discrimination of MRSA strains.

Diagnostic Value of the Amplicor PCR Assay for Initial Diagnosis and Assessment of Treatment Response for Pulmonary Tuberculosis

We evaluated the Amplicor PCR assay as an initial diagnostic tool on the basis of clinical diagnosis, and assessed this assay as a follow‐up test for patients with pulmonary tuberculosis during chemotherapy. Of the 208 specimens from 155 patients who were bacteriologically and/or clinically diagnosed with active tuberculosis before chemotherapy, 144 were Amplicor PCR‐positive (sensitivity, 69.2%), which was equal to the results of culturing. Among 89 specimens which showed positive results by smear and culturing, the Amplicor PCR assay detected 87 (97.8%), whereas among 55 specimens which showed smear‐negative but culture‐positive results, the Amplicor PCR assay detected 46 (83.6%)(P = 0.003). No false positive results were found in the two systems (specificity, 100%, 120/120). The Amplicor PCR assay was also evaluated as a follow‐up test using 926 specimens from 207 patients receiving active tuberculosis chemotherapy. Among 433 specimens which showed Amplicor‐PCR positive, 222 (51.3%) were culture‐negative. On the other hand, among 233 culture‐positive specimens, only 12 (5.2%) were Amplicor PCR‐negative. Therefore, this assay is useful for the rapid diagnosis of tuberculosis. The duration of Amplicor PCR‐positive after culture‐negative conversion was significantly associated with the presence of cavitary lesion, smear‐positive specimens before treatment, and smear‐positive specimens with negative cultures during chemotherapy.

Carotenoid pigments of genus rhodococcus

A study of carotenoid pigments of the genus Rhodococcus was carried out. According to carotenes contained, Rhodococcus species were divided into three groups: the first group of Rhodococcus luteus, R. coprophilus, R. lentifragmentus, and R. maris, which formed β‐carotene; the second group of R. equi, R. rubropertinctus, R. aichiensis, R. sputi, R. chubuensis, R. obuensis, R. bronchialis, R. roseus, R. rhodochrous, R. rhodnii, and R. terrae, which formed γ‐carotene‐like substance; and the third group of R. aurantiacus, which formed neither carotene. Other carotenoid pigments were different according to the species.

Heterogeneity of Phenotypic and Genotypic Traits Including Organic-Acid Resistance in Escherichia coli O157 Isolates

We undertook an epidemiologic study for the sensitivity of both Shiga‐like toxin (Slt)‐producing Escherichia coli (STEC) O157 and non‐STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic‐acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.

Types of methicillin-resistant Staphylococcus aureus associated with high mortality in patients with bacteremia

Forty-seven strains of methicillin-resistantStaphylococcus aureus (MRSA) isolated from 47 patients with bacteremia were analyzed by chromosomal DNA digestion pattern using pulsed-field gel electrophoresis and evaluated for serological coagulase type, enterotoxin type, and toxic shock syndrome toxin-1 production. The mortality rate was significantly higher in the older patients (≥51 years of age) than in the younger patients (≤50 years of age) (50% vs. 4%, p=0.0007). Methicillin-resistantStaphylococcus aureus strains of serological coagulase type II were more likely to be associated with mortality in older patients than were strains of the other types (p=0.037).

Campylobacter fetus subsp. fetus Cholecystitis in a Patient with Advanced Hepatocellular Carcinoma

Acute cholecystitis due to Campylobacter fetus subsp. fetus is very uncommon. We report a case of cholecystitis and obstructive jaundice in which cultured bile grew this organism. The patient had a 4-year history of hepatocellular carcinoma, resulting in common bile duct obstruction due to abdominal lymph node metastasis. Microscopic examination of her bile showed multiple Gram-negative curved organisms and C. fetus subsp. fetus was isolated under microaerophilic conditions. Therefore, we should be aware of this organism and use microaerophilic culture in association with the result of microscopic examination of bile specimens.

Polymerase chain reaction for the differentiation of Mycobacterium intracellulare and Mycobacterium avium

Polymerase chain reaction (PCR) amplification was performed using DNA purified from 15 mycobacterial type strains and from 21 specimens isolated from patients suspected to have non-tuberculous mycobacterial diseases. Using a primer set of MTB1-MTB2, 11 specimens out of 21 were Mycobacterium avium and 8 were M. intracellulare, which were verified by the Gen Probe Rapid Diagnostic System for the M. avium complex (MAC). One of the remaining 2 specimens which did not hybridize with the probe for the MAC was identified as M. kansasii and the other was not specifically identified by the conventional culture method. PCR amplification, using a primer set of TB1-TB3, was also performed for the specific identification of M. tuberculosis complex.

Relationship between mycobacterial species and their carotenoid pigments

A study of the relationship between mycobacterial species and their carotenoid pigments was carried out. According to the carotenoid pigments contained, the mycobacterial species tested were divided into four groups: the first group of Mycobacterium kansasii and M. marinum, which formed principally only β‐carotene; the second group of M. gordonae, M. scrofulaceum, M. szulgai, M. xenopi, M. flavescens, M. phlei, M. rhodesiae, M. neoaurum, and M. aichiense, which formed β‐carotene and a zeaxanthin‐like substance; the third group of M. aurum and M. obuense, which formed β‐carotene and an eschscholtzxanthin‐like substance; and the fourth group of M. chubuense and M. tokaiense, which formed β‐carotene and zeaxanthin‐ and eschscholtzxanthin‐like substances. The common carotenoid pigment throughout the genus Mycobacterium was β‐carotene and the hypophasic carotenoids differed according to the species.