Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae

Abstract

Macrolide resistance in Streptococcus pneumoniae emerged in the U.S. and globally during the early 1990's. The RNA methylase encoded by erm(B) and the macrolide efflux genes mef(E) and mel were identified as the resistance determining factors. These genes are disseminated in the pneumococcus on mobile, often chimeric elements consisting of multiple smaller elements. To better understand the variety of elements encoding macrolide resistance and how they have evolved in the pre- and post-conjugate vaccine eras, the genomes of 121 invasive and ten carriage isolates from Atlanta from 1994 to 2011 were analyzed for mobile elements involved in the dissemination of macrolide resistance. The isolates were selected to provide broad coverage of the genetic variability of antibiotic resistant pneumococci and included 100 invasive isolates resistant to macrolides. Tn916-like elements carrying mef(E) and mel on the Macrolide Genetic Assembly (Mega) and erm(B) on the erm(B) element and Tn917 were integrated into the pneumococcal chromosome backbone and into larger Tn5253-like composite elements. The results reported here include identification of novel insertion sites for Mega and characterization of the insertion sites of Tn916-like elements in the pneumococcal chromosome and in larger composite elements. The data indicate that integration of elements by conjugation was infrequent compared to recombination. Thus, it appears that conjugative mobile elements allow the pneumococcus to acquire DNA from distantly related bacteria, but once integrated into a pneumococcal genome, transformation and recombination is the primary mechanism for transmission of novel DNA throughout the pneumococcal population.

Keywords: Streptococcus pneumoniae; antibiotic resistance; integrative and conjugative elements; macrolides; mobile genetic elements; transposons.

Figure 1

Macrolide resistance determinants associated with mobile elements in pneumococci. Conjugative transposon Tn916 and Tn916-like mosaic elements carrying Mega (blue), and erm(B) on the erm(B) element and Tn917 (purple). The yellow arrows indicate tet(M) encoding tetracycline resistance. Black arrows indicate insertion sites.

Figure 2

Whole genome SNP-derived phylogenetic tree. Whole genome single nucleotide polymorphism analysis of 166 genomes, including 147 from the present study and nine publically available closed genomes, using the TIGR4 genome as reference. The tree was rooted using S. infantis SPAR10 as the outgroup. Numbers on the perimeter indicate the MLST clonal complex of the isolates and are colored the same as the branches leading to the isolates in each complex. The clonal complex of each sequence type was defined as the founder ST of a clonal group, as predicted by eBURST analyses of the entire S. pneumoniae MLST databased accessed in September, 2014 (http://pubmlst.org/spneumoniae/). Isolate names and serotype labels colored blue indicates that the strain was isolated prior to the introduction of PCV7 in 2000. Red colored names and serotypes indicate the strains were isolated in 2000 or later. Colored dots indicate a Mega insertion in the isolate and are color coded by Mega insertion site, light blue, class I; dark blue, class II; red, class III; pink, class IV; green, class V (Tn916 orf6); and gold, novel insertion site.

Figure 3

Mega insertion sites. Target sites of the mobile element Mega encoding macrolide efflux genes mef(E) and mel. Putative six base coupling sequences (C.S.) are shown adjacent to both ends of Mega (italics). Underlined letters indicate conserved nucleotides that may be the consensus recognition sequence 5′ TTTCCNCAA 3′. Class IV was composed of three distinct, subclasses, IVa, IVb, and IVc. Class IVb and IVa share the same left junction and IVa and IVc same right junction (shaded).

Figure 4

Variations of the Pneumococcal Pathogenicity Island-1 associated with Mega. The Pneumococcal Pathogenicity Island-1 in TIGR4 extends from rumA (SP_1029) to ftsW (SP_1065). Strain NP112 is closely related to GA16242 and GA17545, the Mega-2.IVa and IVc strains, but contains the conserved region of PPI-1. Red blocks represent regions of homology. Arrows indicate genes. Green, PPI-1 borders; yellow, iron uptake operon piaABCD; purple, osmotic stress tolerance operon phgABC; gray, PPI-1 conserved region genes not otherwise labeled; white, PPI-1 variable region genes; blue, Mega; orange, ISSmi1. The light blue double arrow indicates the presence of an undefined transposon-like sequence from S. equi subspecies zooepidemicus.

Figure 5

Insertion sites of Tn916-like elements. Insertion sites within composite elements. The terminal sequences of Tn916 are shown in italics. Coupling sequences are underlined. Plain text indicates the sequences of ICE flanking the Tn916-like element. (R) indicates that the Tn916-like element is in the reverse orientation. The ORFs that flank the Tn916-like elements are indicated adjacent to the ICE sequences on either side.

Figure 6

Comparison of ICESp23FST81-like elements encoding macrolide resistance. The location of the Tn916-like element in each is indicated by the Tn5253 genes shown as arrows above each track. Green arrows, Tn916-genes; Blue arrows, mef(E) and mel; yellow arrows, tet(M); pink arrows, integrated plasmid pC194; red arrows, cat.