A total of 1,104 strains of Streptococcus pneumoniae were isolated from hospitalized patients (75.5%) and from colonization sites at the Public Health Institute (24.5%) during the last 4 years (1989-1993). Penicillin resistance (defined as MIC > or = 0.1 microgram/liter) was found to increase in time: 4.6% in 1989, 4.9% in 1990, 6.4% in 1991, 11.3% in 1992, and 17% in 1993. In 1992, 0.8% of the strains had penicillin MIC = 2 mg/liter. Increase in penicillin resistance was also evident among invasive strains [blood, cerebrospinal fluid (CSF), pleura]: 2/92 isolates from 1989-1992 (2.3%) were penicillin resistant as compared to 7/101 isolates from 1992 (6.9%). Multiresistance (resistance to three or more antibiotics) was 4.4% with penicillin, tetracycline, chloramphenicol being the predominant resistant phenotype at the end of 1992. In 1993, strains with resistance to penicillin plus chloramphenicol plus tetracycline increased further. Erythromycin resistance was usually associated with clindamycin resistance, but the majority of such strains remained susceptible to penicillin. Drift in the direction of increasing MIC values was also noted during this 3-year period among "susceptible" isolates: bacteria with MIC of 6-10 ng/ml representing the majority of isolates in 1990-1991 decreased and were replaced by cells with MIC of 20 ng/ml. The most frequent pneumococcal serotypes were 23, 19, 3, 6, 14, 9, and 8, with the frequency of type 9 increasing from 2.9% (in 1989) through 3.2% (1990) to 6.4% (1992). Among all antibiotic resistant strains, the prevalent serotypes were 23, 9, 14, and 6. The chromosomal backgrounds of resistant isolates tested by pulsed-field gel electrophoresis showed close resemblance to the background of the serotype 23F multiresistant clone first identified in Spain. The data suggest that the recent large increase in the frequency of penicillin-resistant pneumococci represents the import of this clone into Portugal during the early 1990s. The increased frequency of drug-resistant pneumococci highlights the importance of continued surveillance of these dangerous pathogens.
We studied the genetic relatedness of 47 Portuguese penicillin-resistant 9V and 23F Streptococcus pneumoniae of clinical origin, using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism analysis. PFGE fingerprinting showed that 24 isolates of serotype 9V and 23 isolates of serotype 23F were variants of the Spain(9V)-3 and Spain(23F)-1 clones, respectively. Fingerprinting of pbp1a, pbp2b and pbp2x genes showed that all the penicillin-resistant clones gave similar HinfI restriction patterns. In this study, serotypes 9V and 23F have different clonal origins and identical PBP genotypes, suggesting a horizontal transfer of resistance. Visual and computer-assisted analysis of PFGE patterns correlated well (r = 0.983).
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