The aim of this study was to research the distribution in the Kinki region of Japan of Enterobacteriaceae and Pseudomonas aeruginosa that produce extended-spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL). One thousand isolates, 200 of each of four enterobacterial species (i.e. Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens) and 200 of P. aeruginosa, were collected from seven different laboratories during two 2 month periods, one in 1998 and one in 2000. A double-disc synergy test (DDST) and 2-mercaptopropionic acid inhibition test (2-MPAT) were used to confirm beta-lactamase-producing isolates. The DDST was positive for one isolate of E. coli, five of K. pneumoniae, two of E. cloacae and 14 of S. marcescens. The 2-MPAT was positive for five isolates of S. marcescens and two of P. aeruginosa. We identified the beta-lactamase type of each isolate by molecular confirmatory tests (isoelectric focusing, PCR and DNA sequencing): CTX-M-3 ESBLs (three isolates of K. pneumoniae, two of E. cloacae and 13 of S. marcescens), CTX-M-2 ESBL (one isolate of K. pneumoniae), SHV-12 ESBLs (one isolate of E. coli and one of S. marcescens), CTX-M-3 and SHV-12 combination ESBL (one isolate of K. pneumoniae) and IMP-1 MBLs (five isolates of S. marcescens and two of P. aeruginosa). In conclusion, many species of Gram-negative bacilli that produce CTX-M-3 ESBLs and IMP-1 MBLs were disseminated widely in different hospitals of the Kinki region of Japan. Therefore, monitoring of laboratory bacterial ecology seems important to stop the spread of these strains through nosocomial outbreaks.
Two novel actinobacteria, designated IFM 12276T and IFM 12275, were isolated from clinical specimens in Japan, and their taxonomic positions were investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strains IFM 12276 T and IFM 12275 have completely identical 16S rRNA gene sequences and were closely related to members of the genus Nocardia . The highest 16S rRNA gene sequence similarity was observed to Nocardia beijingensis (99.6 %) and Nocarida sputi (99.6 %), followed by Nocardia niwae (99.3 %) and Nocardia araoensis (99.3 %). The whole-cell hydrolysates of strains IFM 12276T and IFM 12275 contained meso-diaminopimelic acid, arabinose and galactose. The acyl type of muramic acid was N-glycolyl. The predominant isoprenoid quinone was MK-8(H4, ω-cycl.) and the principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. Strains IFM 12276T and IFM 12275 contained mycolic acids that co-migrated with those from the type strain of N. niwae . These chemotaxonomic features corresponded to those of the genus Nocardia . Meanwhile, the differences in some phenotypic characteristics, along with the results of average nucleotide identity and digital DNA–DNA hybridization analyses, indicated that strains IFM 12276 T and IFM 12275 should be distinguished from the recognized species of the genus Nocardia . Therefore, these strains represent a novel species of the genus Nocardia , for which the name Nocardia sputorum sp. nov. is proposed. The type strain is IFM 12276T (=NBRC 115477T=TBRC 17096T).
We studied the susceptibility to penicillin G (PCG) and other antimicrobiotics in 235 clinical isolates of Streptococcus pneumoniae. Samples were collected between April 1 and June 30, 2000 from nine medical institutions of the Kinki Region of Japan. We classified the minimum inhibitory concentration (MIC) of PCG according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria. The overall rate of all types of S. pneumoniae resistance was 53.2% (penicillin-susceptible S. pneumoniae (PSSP): 46.8%, penicillin-intermediate S. pneumoniae (PISP): 42.6%, penicillin-resistant S. pneumoniae (PRSP): 10.6%). In other antimicrobiotics, the resistance (R)/intermediate susceptibility (I) rates (R/I%) were as follows: ceftriaxone, 28.9%; cefotaxime, 7.7%; imipenem, 8.9%; meropenem, 9.8%; clarithromycin, 82.6%; clindamycin, 42.1%; levofloxacin, 0.4%; vancomycin, 0%. We used the polymerase chain reaction to study the mutations of the penicillin-binding proteins pbp1 a, pbp2b, and pbp2x in 140 strains of S. pneumoniae in the MIC for PCG was < 0.5 microgram/ml. Among the 109 strains of PSSP, 32 (29.4%) had no mutation and 77 (70.6%) showed mutation of more than one of the pbp mutations. Among the 31 strains of PISP, only 1 strain (3.2%) was not mutated. Since 70.6% of the strains classified as PSSP had pbp mutations, S. pneumoniae clearly can acquire resistance to anti-microbiotics. In the future, a comprehensive surveillance of S. pneumoniae is necessary.
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