A mosaic tetracycline resistance gene tet(S/M) detected in an MDR pneumococcal CC230 lineage that underwent capsular switching in South Africa

Abstract

Objectives: We reported tet(S/M) in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions.

Methods: We whole-genome sequenced 12 254 pneumococcal isolates from 29 countries on an Illumina HiSeq sequencer. Serotype, multilocus ST and antibiotic resistance were inferred from genomes. An SNP tree was built using Gubbins. Temporal spread was reconstructed using a birth-death model.

Results: We identified tet(S/M) in 131 pneumococcal isolates and none carried other known tet genes. Tetracycline susceptibility testing results were available for 121 tet(S/M)-positive isolates and all were resistant. A majority (74%) of tet(S/M)-positive isolates were from South Africa and caused invasive diseases among young children (59% HIV positive, where HIV status was available). All but two tet(S/M)-positive isolates belonged to clonal complex (CC) 230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sublineage predicted to exhibit resistance to penicillin, co-trimoxazole, erythromycin and tetracycline. The birth-death model detected an unrecognized outbreak of this sublineage in South Africa between 2000 and 2004 with expected secondary infections (effective reproductive number, R) of ∼2.5. R declined to ∼1.0 in 2005 and <1.0 in 2012. The declining epidemic could be related to improved access to ART in 2004 and introduction of pneumococcal conjugate vaccine (PCV) in 2009. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sublineage.

Conclusions: The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognized outbreak of CC230 in South Africa. Capsular switching in this MDR sublineage highlighted its potential to continue to cause disease in the post-PCV13 era.

Figure 1.

Schematic representation of the mosaic structure of tet(S/M) alleles of the current and previous studies. The bars in grey and in white indicate amino acid sequences with high identity to Tet(S) and Tet(M), respectively. The reference sequences for tet(M) and tet(S) were retrieved from NCBI GenBank using accession numbers M85225 and FN555436, respectively.

Figure 2.

An SNP tree constructed with CC230 tet(S/M)-positive isolates (n=129) and tet(S/M)-negative carriage/disease isolates (n=260) collected from 20 countries. The tree was built based on 13 405 SNPs extracted from an alignment outside recombination regions, created by mapping reads of each isolate to the sequence of an ST230 reference strain, PMEN global clone Denmark¹⁴-32, PMEN32 (ENA accession number ERS1706837). Penicillin resistance was predicted based on the pbp1a, pbp2x and pbp2b sequences;^, tetracycline and erythromycin resistance were predicted based on the presence of tet(M), tet(O) and tet(S/M), and erm(B) and mef(A), respectively. Co-trimoxazole resistance was predicted based on the presence of mutation I100L in folA and any indel within amino acid residues 56–67 in folP, while the presence of either mutation was predicted to confer a co-trimoxazole-intermediate phenotype.

Figure 3.

(a) Malawi, Mozambique and administration regions of South Africa. (b) Timed phylogeny for S. pneumoniae tet(S/M) CC230 sublineage (n=129) reconstructed using BEAST. Tree branches are coloured according to the geographical locations in (a), except for the branch for an isolate collected from the USA, coloured in brown. (c) Vaccine serotype 14 is indicated in blue, whereas non-vaccine serotype 23A is indicated in orange.

Figure 4.

(a) Birth–death skyline plot of inferred changes in R of S. pneumoniae tet(S/M) CC230 sublineage using IPD isolates (n=105). (b) Coalescent-based skyline plot of inferred changes in the effective population size (Ne) of the S. pneumoniae tet(S/M) CC230 sublineage using both IPD and carriage isolates (n=129) and (c) using only IPD isolates (n=105). The black continuous line shows the median of R in (a) and Ne in (b) and (c). The background area represents the 95% HPD intervals. R>1 indicates a growing epidemic, whereas R<1 indicates a declining epidemic.

Figure 5.

Comparison of ICE identified in CC230 with Spain23^F-1 (PMEN1). Grey bands between the sequences indicate BLASTN matches.