Molecular evolution in a multidrug-resistant lineage of Streptococcus pneumoniae: emergence of strains belonging to the serotype 6B Icelandic clone that lost antibiotic resistance traits

Abstract

Since their first detection in 1988, penicillin-resistant Streptococcus pneumoniae isolates have rapidly spread in Iceland to account for close to 20% of all pneumococcal disease in that country by 1993. The major component (70%) of the resistant pneumococci identified from 1989 to 1992 was the progeny of a single multidrug-resistant clone (Icelandic clone) with a homogeneous chromosomal macrorestriction profile and identical multilocus enzyme type expressing serotype 6B and resistance to penicillin, tetracycline, chloramphenicol, erythromycin, and trimethoprim-sulfamethoxazole. The rest of the non-penicillin-susceptible isolates included bacteria with serotype 6A and serogroups 19 and 23. The unique geographic and epidemiological setting and the availability of a complete collection of all non-penicillin-susceptible isolates of S. pneumoniae in Iceland prompted us to carry out a molecular epidemiological study to monitor the fate of the Icelandic clone between 1989 and 1996; in addition, we wished to extend the characterization to representative groups of all non-penicillin-susceptible serotype 6B pneumococci which showed variations in antibiotype and which were recovered in Iceland between late 1989 and the end of 1996. Also included in the study were non-penicillin-susceptible isolates of serogroup 23. Pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA and Southern hybridization with the lytA DNA probe and probes specific for antibiotic resistance genes were used to characterize pneumococcal isolates. The results show that (i) the Icelandic clone remained the predominant type among penicillin-resistant S. pneumoniae through 1996; (ii) the emergence of variants of the Icelandic clone which had lost one or more of the antibiotic resistance phenotypes and/or resistant genes, singly or in combination, was documented during the surveillance period; and (iii) isolates belonging to the internationally spread multidrug-resistant serotype 23F clone were present in the Icelandic collection since late 1989 but did not increase in number during the subsequent years.

FIG. 1

PFGE (A) and Southern blotting (B and C) of isolates characteristic of the Spanish/Icelandic clone. SmaI digests were separated by PFGE and subsequently transferred to nylon membranes for hybridization. (A) Lanes 3 through 14 show the typical PFGE pattern of the Spanish/Icelandic clone (pattern A1). The two largest fragments cannot be separated under these conditions. Lanes 15 through 18 show isolates with a deletion in one of the two largest fragments which, subsequently, show up in the PFGE pattern (pattern A2). Lanes 19 and 20 show patterns A3 and A4, respectively. Lanes 1, 2, 21, and 22 are molecular size markers. (B) Southern blot of the gel in panel A with a tetM probe. Isolates with PFGE patterns A2 and A3 show no signal with the probe. (C) a Southern blot of the gel in panel A with an ermB probe. As in panel B, isolates with PFGE patterns A2 and A3 show no signal with the probe.

FIG. 2

Evolution of the Spanish/Icelandic clone in Iceland. The majority of the strains belonging to the Spanish/Icelandic clone appear unchanged and are represented by the bold vertical arrow. The appearance of variants that have lost the resistance phenotype is represented by the horizontal arrows, and the vertical arrows demonstrate how many isolates of each variant have been detected (from 1989 through September 1996) and to which group they belong. The boxes represent (from left to right) resistance (R) or susceptibility (S) to penicillin (P), tetracycline (T), erythromycin (E), and chloramphenicol (C).

FIG. 3

PFGE of isolates of serotype 23F. Lane 4 shows the pattern of a control strain of the Spanish/Cleveland clone of serotype 23F. Lanes 5 through 8 are Icelandic isolates of serotype 23F, identical to the Spanish/Cleveland clone. Lane 3 and lanes 9 through 13 show the dominant PFGE patterns of serotype 23F isolates in Iceland. Lanes 14 through 17 show the patterns of other isolates of serotype 23F in Iceland. Lanes 1, 2, 18, and 19 are molecular size markers.