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We have developed a modified cyclodextrin solid (MCS) medium using the selective antibiotic cefdinir. MCS medium exhibited higher sensitivity (95.6%; any culture-positive sample as reference) and greater inhibition of nasopharyngeal flora than did Bordet-Gengou agar (65.2%, P = 0.009) or cyclodextrin solid medium (39.1%, P < 0.001).

Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new neonatal disease caused by toxic shock syndrome toxin 1 (TSST-1). We conducted a prospective surveillance study and characterized the methicillin-resistantStaphylococcus aureus(MRSA) strains isolated from patients with NTED and compared them with the strains from patients with other MRSA infections and asymptomatic carriers. The study was performed in the neonatal intensive care unit and a general neonatal and maternal ward in the Tokyo Women's Medical University Hospital (TWMUH) from September to December 1998. Among 103 patients eligible for the study, MRSA was detected in 62 (60.2%) newborns; of these 62 newborns, 8 (12.9%) developed NTED, 1 (1.6%) had another MRSA infection, and 53 (85.5%) were asymptomatic MRSA carriers. Sixty-nine MRSA strains were obtained from the 62 newborns. DNA fingerprinting by pulsed-field gel electrophoresis showed two clusters: clone A with 8 subtypes and clone B. Sixty-seven of the 69 MRSA strains (97.1%) belonged to clone A, and type A1 was the most predominant (42 of 69 strains; 60.9%) in every neonatal and perinatal ward. All but one of the clone A strains had the TSST-1 and staphylococcal enterotoxin C genes. We also analyzed eight MRSA strains from eight NTED patients in five hospitals in Japan other than TWMUH. All the MRSA strains from NTED patients also belonged to clone A. These results suggest that a single clone that predominated in the neonatal wards of six hospitals might have caused NTED. However, the occurrence of NTED might not be dependent on the presence of an NTED-specific strain.

Metallo-β-lactamases (MBLs) are transmissible carbapenemases of increasing prevalence in Gram-negative bacteria among health care facilities worldwide. Control of the further spread of these carbapenem-resistant bacteria relies on clinical microbiological laboratories correctly identifying and classifying the MBLs. In this study, we evaluated a simple and rapid method for detecting IMP, the most prevalent MBL in Japan. We used an immunochromatography (IC) assay for 181 carbapenem-nonsusceptible (CNS) (nonsusceptible to imipenem or meropenem) strains comprising 74 IMP-producing and 33 non-IMP-producing strains of non-glucose-fermenting Gram-negative rods (NFGNR), as well as 64 IMP-producing and 10 non-IMP-producing Enterobacteriaceae strains. The IC assay results were compared to those from the double-disk synergy test (DDST), the MBL Etest, and the modified Hodge test (MHT) (only for Enterobacteriaceae ). The IMP type was confirmed by specific PCR and direct sequencing. The IC assay detected all of the IMP-type MBLs, including IMP-1, -2, -6, -7, -10, -11, -19, -20, and -22 and IMP-40, -41, and -42 (new types), with 100% specificity and sensitivity against all strains tested. Although the sensitivity and specificity values for the DDST and MHT were equivalent to those for the IC assay, the MBL Etest was positive for only 87% of NFGNR and 31% of Enterobacteriaceae due to the low MIC of imipenem, causing an indeterminate evaluation. These results indicated that the IC assay might be a useful alternative to PCR for IMP MBL detection screening.

The rapid ID 32 Strep system (bioMérieux, Marcy l'Etoile, France) was evaluated for its ability to identify 21 species of viridans group streptococci; results were compared with DNA-DNA hybridization results and results of conventional physiological tests. A total of 171 strains of the 21 species including 147 clinical strains was analyzed. Of the 156 strains of species included in the database of this system, 136 strains (87%) were correctly identified. Incorrect identification occurred for 13 strains (8%), and no identification was given for 7 strains (5%). It was difficult to differentiate S. mitis and S. oralis accurately with this system. Of the 17 strains identified as S. mitis by the rapid ID 32 Strep system, the results of DNA-DNA hybridization were in agreement for only 3 strains. S. crista and S. parasanguis, which are not included in the database, were identified as S. mitis or S. sanguis or were not identified, but S. parasanguis could probably be identified by using the rapid ID 32 Strep system because the biochemical profile is well characterized for this species. The rapid ID 32 Strep system can be used to differentiate most species for which phenotypic characteristics have been described if the database is revised according to recently reported amended criteria for the identification of viridans group streptococci. However, identification of a few species such as S. mitis and S. oralis is problematic with this system.

In clinical microbiology, bacterial identification is labor-intensive and time-consuming. A solution for this problem is the use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). In this study, we evaluated a modified protein extraction method of identification performed on target plates (on-plate extraction method) with MALDI-TOF (Bruker Microflex LT with Biotyper version 3.0) and compared it to 2 previously described methods: the direct colony method and a standard protein extraction method (standard extraction method). We evaluated the species of 273 clinical strains and 14 reference strains of staphylococci. All isolates were characterized using the superoxide dismutase A sequence as a reference. For the species identification, the on-plate, standard extraction, and direct colony methods identified 257 isolates (89.5%), 232 isolates (80.8%), and 173 isolates (60.2%), respectively, with statistically significant differences among the three methods ( P < 0.05). In conclusion, the on-plate extraction method is at least as good as standard extraction in identification rate and has the advantage of a shorter processing time.

Mycobacterium haemophilum is an emerging opportunistic pathogen, and since 1989, infections caused by this organism have been identified more frequently in the New York City area than in any other region of the United States. A DNA fingerprinting method, based on restriction fragment length polymorphisms (RFLPs) was developed. A genomic library of M. haemophilum isolate 1A was constructed; screening the library yielded a recombinant strain that incorporated a genetic element present in multiple copies in the M. haemophilum genome. This clone was used to produce a probe for RFLP analyses of PvuII digests of genomic DNA. We used this probe to determine the RFLP patterns of 43 clinical isolates of M. haemophilum from 28 patients. A total of six distinct patterns were observed. Two patterns, designated types 1 and 2, accounted for 91% of the infections in patients from the New York City area. Two isolates from Arizona had identical patterns but were distinct from those of New York isolates, and an isolate from Israel, the type strain, had another distinct pattern (type 6). The type 6 pattern was also seen in a recent isolate from Norway. All of the type 1 isolates and 60% of the type 2 isolates were recovered from patients with AIDS in the New York City area. This molecular subtyping method should provide a useful tool for epidemiological studies and may help identify the associated risk factors, vehicles, and possible reservoirs of this newly emerging pathogen.

We surveyed methicillin-resistant coagulase-positive staphylococcus (MRCPS) strains from 57 (26 inpatient and 31 outpatient) dogs and 20 veterinary staff in a veterinary teaching hospital. From the staff, three MRCPS strains were isolated, and two were methicillin-resistant Staphylococcus aureus (MRSA). In contrast, 18 MRCPS strains were detected in both inpatient (12 of 26 [46.2%]) and outpatient (6 of 31 [19.4%]) dogs. Among them, only one strain was MRSA. Using direct sequencing of sodA and hsp60 genes, the 18 MRCPS strains other than MRSA from a staff and 17 dogs, were finally identified as Staphylococcus pseudintermedius , a novel species of Staphylococcus from a cat. All of the methicillin-resistant S. pseudintermedius (MRSP) strains were multidrug resistant to erythromycin, clindamycin, trimethoprim-sulfamethoxazole, and levofloxacin. Most of the MRSP strains showed high-level resistance to oxacillin (≥128 μg/ml, 15 of 18 [83.3%]), and 10 of 15 (66.7%) high-level oxacillin-resistant MRSP strains carried type III SCC mec . DNA fingerprinting of MRSP strains by pulsed-field gel electrophoresis yielded eight clusters: clone A with four subtypes, clone B with four subtypes, clone C with three subtypes, and five other different single clones. MRSP strains from the staff and some inpatient and outpatient dogs shared three major clones (clones A, B, and C), but the strains of the other five different clusters were distributed independently among inpatient or outpatient dogs. This genetic diversity suggested that the MRSP strains were not only acquired in this veterinary teaching hospital but also acquired in primary veterinary clinics in the community. To our knowledge, this is the first report of MRSP in dogs and humans in a veterinary institution.

The minimum growth-inhibitory concentrations (MICs) of azole antifungals were compared to their minimum sterol 14α-demethylation-inhibitory concentrations (MDICs) for clinical fungal isolates. The ascomycetous Candida yeasts tested were clearly divided into two groups: group I, consisting of C. albicans , C. tropicalis , and C. lusitaniae , had MICs that were much higher than the MDICs, whereas group II, comprising C. glabrata , C. parapsilosis , C. guilliermondii , and C. krusei , had MICs that were approximately equal to the MDICs. In the ascomycetous fungi Aspergillus fumigatus and Sporothrix schenckii , the MICs were indistinguishable from the MDICs. In the basidiomycetous fungi Cryptococcus ( Filobasidiella ) neoformans , C. curvatus , and Trichosporon asahii , the MICs and the MDICs were practically identical. These results support the notion that there are two distinct classes of fungi differing in their degree of tolerance to sterol 14α-demethylation deficiency. These findings have significant implications for both fungal physiology and antifungal chemotherapy.