Clonal success of piliated penicillin nonsusceptible pneumococci

Abstract

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain(9V)-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000-2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall approximately 50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet.

Fig. 1.

Global spread of PNSP of ST156 and related clones. PNSP related to ST156 have spread from Spain to most continents. In Europe they have been found in several countries, such as Austria, Belgium, the Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, The Netherlands, Norway, Poland, Portugal, Spain, Sweden, Switzerland, and the U.K. The first year of isolation as reported in the MLST database is written in parentheses. TLV, triple locus variants of ST156.

Fig. 2.

ST156 among dominant PNSP of types 9V, 14, and 19F in Sweden. (A) Frequency of serotypes 9V, 14 and 19F among pneumococci with reduced susceptibility to penicillin (PNSP) in Sweden. (B) Percentage of the clonal cluster of ST156 (SLV and DLV included) among PNSP of types 9V, 14 and 19F, respectively, in Sweden. Type 9V has been estimated based on molecular typing of 80 strains of type 9V.

Fig. 3.

Differences in gene content between ST156 and non-ST156 isolates. (A) The 55 most statistically significant genes that differed between four pneumococcal isolates of ST156 (of serotype 9V and 14) and two isolates of ST239 (of serotype 9V) and ST709 (of serotype 14), respectively (SI Table 2) are shown. Also, differences between two 19F isolates of ST156 and one 19F isolate of ST425 are shown. Capsular genes are not included. Genes encoding the rlrA islet are marked in gray. (B) Western blot analysis of pneumococcal isolates belonging to serotypes 9V, 14, and 19F with ST156 or with three nonrelated STs using antibodies to the pilus protein RrgB. The extracts of strains BHN99 and BHN98 were concentrated 26 times by precipitation by trichloric acid. (C) Differences between isolates of serotypes 9V, 14, and 19F belonging to the same clone, ST156, are shown. Yellow indicates that the gene is present, dark blue indicates that it is absent (P < 0.01), and light blue indicates that it is likely to be absent but not statistically significant. Capsular genes are not included. Genes encoding for rlrA islet are marked in gray.

Fig. 4.

Genetic differences between capsular switch variants of ST156 localize to a 750-kb region. A schematic overview of the TIGR4 genome in which differences between capsular switch variants of ST156 are shown within a 750-kb region centered on the capsular locus. Marked in black are the genes differing between the different isolates of ST156. Marked in gray are genes encoding the capsule. A star indicates the position of the closest TIGR4 genes to the R6 specific region, which differs between the ST156 isolates.

Fig. 5.

A piliated 19F isolate outcompetes a nonpiliated 19F isolate in a murine colonization model. Competitive index calculated after intranasal challenge of 12 mice with equal amounts of the clinical isolates BHN99 and BHN98, both belonging to serotype 19F and clone ST156, but differing in the presence of the pilus gene cluster. The competitive index in nasopharyngeal lavages was calculated after 7 days. The piliated isolate BHN99 was shown to outcompete the nonpiliated isolate BHN98 because the competitive index was <1.