Heterogeneity of Tn5253-Like Composite Elements in Clinical Streptococcus pneumoniae Isolates

Abstract

Several drug resistances in Streptococcus pneumoniae are associated with mobile genetic elements, which are loosely subdivided into a group of smaller (18- to 27-kb) and a group of larger (>50-kb) elements. While the elements of the former group, which typically carry the tetracycline resistance determinant tet(M) and whose prototype is Tn916 (18 kb), have been studied extensively, the larger elements, whose prototype is Tn5253 (∼65.5 kb), are not as well explored. Tn5253 is a composite structure consisting of two independent conjugative transposons, Tn5251 (which is virtually identical to Tn916) and Tn5252 (∼47.5 kb), with the former inserted into the latter. Tn5252, which so far has only partially been sequenced, carries an integrase gene, driving its site-specific insertion into the host cell genome, and the chloramphenicol resistance cat_pC194 determinant. This study investigated 20 clinical isolates of S. pneumoniae, which were selected on the basis of cat_pC194-mediated chloramphenicol resistance. All 20 isolates harbored a Tn5253-like element. The composite elements (some of which have been completely sequenced) demonstrated considerable heterogeneity that stemmed from a dual variability: in the Tn5252-like element, due primarily to differences in the integrase gene but also to differences in cargo genes and in the overall genetic organization, and in the Tn916-like element, with the possible involvement, besides Tn916, of a number of Tn916 family pneumococcal elements carrying different erythromycin resistance genes. In mating experiments, only one composite element, containing a less typical Tn916 family element, appeared to be nonmobile. Being part of a Tn5253-like composite element may confer on some Tn916-like transposons, which are apparently nontransferable as independent genetic elements, the ability to be mobilized.

A unique recombination-mediated genetic plasticity is a distinctive feature of Streptococcus pneumoniae (3) and a key to its success as a pathogen. In this context, a cause for serious concern is the emergence and increasing spread of multidrug-resistant clinical pneumococci (28), where multiple resistance is generally associated with mobile genetic elements. Although the nomenclature for such elements is evolving, they are often referred to as transposons or as integrative and conjugative elements (ICEs) (21, 33). Pneumococcal mobile elements (which are also found in other streptococci and Gram-positive cocci) can be loosely subdivided into two major groups of smaller (18- to 27-kb) and larger (>50-kb) elements (2, 23).

The smaller genetic elements form the widespread Tn916 family, so called from the prototype, Tn916, an 18-kb transposon originally detected in Enterococcus faecalis when it was still regarded as a Streptococcus species (12). Tn916 family elements typically contain integrase (int₉₁₆) and excisionase (xis₉₁₆) genes and carry the tetracycline (TET) resistance determinant tet(M). These elements have been widely investigated, leading to the discovery of a variety of Tn916 relatives (22). In particular, recent studies have elucidated the actual role and relationships (in terms of structure as well as prevalence) of a number of old and new Tn916-related pneumococcal elements also carrying the erythromycin (ERY) resistance gene erm(B) or mef(E) linked to tet(M) (22, 29).

Conversely, the larger genetic elements have not been investigated quite as extensively. The prototype of this group in S. pneumoniae is Tn5253, an ∼65.5-kb composite structure made up of two independent conjugative transposons, Tn5251 and Tn5252, with the former inserted into the latter (2). Tn5251 (18 kb) is virtually identical to Tn916 in structure and size, with only a few differences occurring, chiefly in the tet(M) gene sequence (19, 25). Tn5252 (∼47.5 kb) is a conjugative transposon containing a chloramphenicol (CHL) resistance gene (cat), namely, the cat_pC194 determinant from the linearized pC194 Staphylococcus aureus plasmid (32), and an integrase gene (int₅₂₅₂) driving its site-specific insertion into the host cell genome (30). Only three Tn5252 regions, representing functional modules, have so far been studied and sequenced: the left terminal (LT) region (accession no. L29324), which contains integration (15), mobilization (26, 27), and UV resistance (18) genes; the conjugal transfer-related (CTR) region (accession no. AF295925) (1); and the right terminal (RT) region (accession no. L29323), which contains the DNA cytosine methyltransferase gene (24). Other genetic and functional properties of Tn5252 are still largely unknown.

Recently, variability of some genes of the Tn5252 LT region in clinical isolates of pneumococci has been reported (14). Moreover, analysis of the complete chromosome sequences of multiresistant S. pneumoniae isolates has disclosed new larger genetic elements, such as ICESp23FST81 (∼81 kb; accession no. FM211187) (8) and Tn2008 (∼67 kb; accession no. CP001033) (11), both carrying cat_pC194 in their Tn5252-like transposon and sharing an identical genome integration site that is different from that of Tn5253.

The present study of 20 isolates, selected on the basis of cat_pC194-mediated CHL resistance, was aimed at extending our knowledge of the presence, genetic organization, and transferability of Tn5253-like composite elements in clinical pneumococci. While all isolates harbored a Tn5253-like element, such elements demonstrated a substantial heterogeneity resulting from combined variabilities in the Tn5252-like and the Tn916-like elements.

MATERIALS AND METHODS

Bacterial strains.

Twenty isolates were selected from a collection of 166 clinical isolates of S. pneumoniae recovered in several Italian laboratories in 2005 to 2008 and representing geographical and chronological diversities. The inclusion criterion was CHL resistance (MIC, ≥8 μg ml⁻¹) mediated by the cat_pC194 gene, determined by PCR using specific primers R3 and F2 (32) (see Table S1 in the supplemental material).

Typing.

Serotyping was performed by the capsular-swelling test using specific antisera (Statens Seruminstitut, Copenhagen, Denmark). Serotypes were indicated with conventional capsular type designations.

Macrorestriction with SmaI endonuclease (Roche Applied Science, Basel, Switzerland) and pulsed-field gel electrophoresis (PFGE) analysis were performed by established methods. If a PFGE pattern was shared by at least two isolates, the type was designated with a capital letter in order of size, as described elsewhere (20).

Antibiotics and susceptibility tests.

CHL, ERY, and TET were purchased from Sigma Chemical Co., St. Louis, MO. MICs were determined by a standard agar dilution method and interpreted according to current breakpoints (4). S. pneumoniae ATCC 49619 was used as a quality control strain.

Amplification experiments.

The principal primers used in this study are listed in Table S1 in the supplemental material. Other primers used to analyze transposon structures and discriminate among Tn916 family pneumococcal elements have been reported elsewhere (5, 6, 10, 17, 29). DNA preparation and amplification and electrophoresis of PCR products were carried out by established procedures and under recommended conditions for the use of the individual primer pairs. The ExTaq system (TaKaRa Bio, Shiga, Japan) was used in amplification experiments expected to yield PCR products exceeding 3 kb in size.

DNA sequence analysis.

All PCR products used for sequence analysis were purified using Montage PCR filter units (Millipore Corporation, Bedford, MA). Amplicons were sequenced (bidirectionally or by primer walking) using ABI Prism (Perkin-Elmer Applied Biosystems, Foster City, CA) with dye-labeled terminators. Sequences were analyzed using the Sequence Navigator software package (Perkin-Elmer Applied Biosystems). Open reading frame (ORF) analysis was performed using the online software ORF finder (http://www.ncbi.nlm.nih.gov/) and NEB cutter V2.0 (New England Biolabs, Ipswich, MA). Sequence similarity and conserved-domain searches were carried out using tools (BLAST and CDART) available online at the National Center for Biotechnology Information of the National Library of Medicine (Bethesda, MD) (http://www.ncbi.nlm.nih.gov).

Conjugation experiments.

Mating assays were performed as described elsewhere (7). Transconjugants were selected on plates containing CHL (5 μg ml⁻¹), in addition to rifampin (10 μg ml⁻¹) and fusidic acid (10 μg ml⁻¹). ERY (1 or 5 μg ml⁻¹) or TET (5 μg ml⁻¹) was also used as a selecting agent with some donors, according to their resistances. S. pneumoniae isolates with different genotypic characteristics were used as donors. Two CHL-, ERY-, and TET-susceptible recipients were used: S. pneumoniae R6RF, a rifampin- and fusidic acid-resistant derivative obtained from the well-known laboratory strain R6, and Streptococcus pyogenes 12RF, a rifampin- and fusidic acid-resistant derivative obtained from a clinical isolate (13).

Nucleotide sequence accession numbers.

The sequences of three new genetic elements, Tn6058, Tn1311, and ICE6094, were submitted to the EMBL/GenBank sequence database and assigned accession no. FM201786, FN667862, and FR670347, respectively.

RESULTS AND DISCUSSION

Characterization of the isolates.

The 20 isolates were characterized for a number of phenotypic and genotypic attributes (Table 1).

TABLE 1.

Phenotypic and genotypic characteristics of the 20 S. pneumoniae isolates^a

Isolate	Serotype	PFGE type	MIC (μg ml−1)			Resistance genes				Tn5253-related composite element
										Tn916 family element	Tn5252-like element genes			Composite element
			CHL	TET	ERY	erm	mef	tet(M)	aphA-3		int5252	xis5252	intSp23FST81
Pn1	19F	OSTb	16	32	0.03	−	−	+	−	Tn916	+	+	−	Tn5253-like
Pn2	35	C	16	16	4	−	mef(E)	+	−	Tn2009	−	−	+	ICESp23FST81-like
Pn3	23A	B	32	64	4	−	mef(E)	+	−	Tn2009	−	−	+	ICESp23FST81-like
Pn4	19F	OST	16	64	>128	erm(B)	−	+	−	Tn6002	+	+	−	Tn6058
Pn5	23F	C	16	32	16	−	mef(E)	+	−	Tn2009	−	−	+	ICESp23FST81-like
Pn6	6B	OST	8	2	>128	erm(B)	−	−	+	SpnRi3erm(B)-like	+	+	−	Tn1311
Pn7	23F	D	16	32	0.125	−	−	+	−	Tn916	−	−	+	ICESp23FST81
Pn8	23F	B	32	64	0.25	−	−	+	−	Tn916	−	−	+	ICESp23FST81
Pn9	3	A	16	16	>128	erm(B)	−	+	−	Tn6002	+	+	−	Tn6058
Pn10	10A	D	16	32	0.03	−	−	+	−	Tn916	−	−	+	ICESp23FST81
Pn11	6B	B	16	64	0.03	−	−	+	−	Tn916	−	−	+	ICESp23FST81
Pn12	9N	OST	16	64	>128	erm(B)	−	+	−	Tn3872	−	−	+	ICE6094
Pn13	23F	OST	16	32	>128	erm(B)	−	+	−	Tn3872	−	−	+	ICESp23FST81-like
Pn14	NTc	OST	16	32	>128	erm(B)	−	+	−	Tn6002	−	−	+	ICESp23FST81-like
Pn15	3	A	16	64	>128	erm(B)	−	+	−	Tn6002	+	+	−	Tn6058
Pn16	19A	OST	16	64	>128	erm(B)	−	+	−	Tn6002	−	−	+	ICESp23FST81-like
Pn17	3	A	16	32	>128	erm(B)	−	+	−	Tn6002	+	+	−	Tn6058
Pn18	NT	OST	32	64	>128	erm(B)	−	+	−	Tn6002	−	−	+	ICESp23FST81-like
Pn19	3	OST	16	32	>128	erm(B)	−	+	−	Tn6002	−	−	+	ICE6094
Pn20	3	A	16	32	>128	erm(B)	−	+	−	Tn6002	+	+	−	Tn6058

^aAll isolates, selected for cat_pC194-mediated CHL resistance, were positive for the integrase gene int₉₁₆ and the excisionase gene xis₉₁₆.

^bOST, one-strain type.

^cNT, nontypeable.

Typing data included serotyping (with 18 isolates falling into nine serotypes and two being nontypeable) and PFGE typing (with the 20 isolates presenting 13 different PFGE patterns). Antibiotic resistance studies indicated that, in addition to CHL, 15 isolates [12 bearing the erm(B) gene and 3 the mef(E) gene] were resistant to ERY and 19 [all bearing the tet(M) gene] were resistant to TET [the only TET-susceptible, tet(M)-negative isolate (Pn6) was also the only one to be positive for the kanamycin resistance gene aphA-3].

All 20 isolates were positive for both the integrase and the excisionase genes of the Tn916 family elements (int₉₁₆ and xis₉₁₆). The particular Tn916-like pneumococcal element involved was identified by amplification assays. The five ERY-susceptible isolates harbored Tn916. Of the 12 erm(B)-positive isolates, 9 harbored Tn6002 (6, 31), 2 Tn3872 (16), and 1 (Pn6) an element resembling a deleted form (with an incomplete macrolide-aminoglycoside-streptothricin [MAS] fragment containing aphA-3 as the sole resistance determinant) of the one previously designated SpnRi3erm(B) (6). All three mef(E)-positive isolates harbored Tn2009 (10).

Comparative analysis of known sequences and amplification experiments to detect Tn5252-like elements.

Comparative analysis of known sequences of the Tn5252 LT, CTR, and RT regions and of genetic elements ICESp23FST81 and Tn2008 showed that the three Tn5252 regions were present and were identical in the two new elements. While their CTR and RT regions had close identities (around 94%) to the corresponding Tn5252 regions, their LT regions differed considerably from the LT prototype, with which they shared only three Tn5252 ORFs (orf4, orf9, and orf10). In particular, the integrase gene of ICESp23FST81 and Tn2008, here designated int_Sp23FST81, was completely different from int₅₂₅₂. Moreover, the latter is adjacent to the excisionase gene xis₅₂₅₂, whereas an excisionase gene is not found when the integrase gene is int_Sp23FST81.

Testing for the two different integrase genes gave a positive PCR for int₅₂₅₂ and xis₅₂₅₂ in 7 isolates [5 harboring Tn6002, 1 Tn916, and 1 the SpnRi3erm(B)-like element] and for int_Sp23FST81 in 13 [4 harboring Tn916, 4 Tn6002, 3 Tn2009, and 2 Tn3872] (Table 1).

Characterization of Tn5253-like composite elements positive for int₅₂₅₂ and xis₅₂₅₂.

In the seven isolates positive for int₅₂₅₂ and xis₅₂₅₂, PCR experiments with suitable primer pairs were performed to understand the Tn5252 structure, seek a linkage with the associated Tn916-like transposon, and elucidate the genome integration site. The results indicated that in all seven isolates the Tn916-like element was inserted into the Tn5252-like element, forming various Tn5253-like composite structures (Table 1), all sharing the specific site of Tn5252 integration into the spr1043 gene of the S. pneumoniae R6 genome (accession no. AE007317) (25, 30). While one isolate (Pn1) bore regular Tn916 and Tn5252 transposons, the other isolates had composite elements where the Tn916 family transposon was Tn6002 (five isolates [Pn4, Pn9, Pn15, Pn17, and Pn20]) or the SpnRi3erm(B)-like element (one isolate [Pn6]). The composite elements from isolates Pn1, Pn20, and Pn6 (representing the three conditions described above) were investigated by amplification and sequencing assays to elucidate their genetic organization and sequence (Fig. 1).

FIG. 1.

Genetic organization and available sequences of the Tn5253-related pneumococcal elements positive for int₅₂₅₂ and xis₅₂₅₂. A black bar representing the genome denotes sequencing analysis; a white bar indicates just amplification analysis (with sequencing limited to particular areas). The CHL resistance determinant cat_pC194 is depicted as a white arrow. Integrase ORFs (int₅₂₅₂ and int₉₁₆) are shown as black arrows. Tn916 family transposons are represented as dotted bars, with resistance genes tet(M) (hatched), erm(B) (checkered), and aphA-3 (spotted) indicated. The three modules of the Tn5252 prototype sequenced so far (LT region, accession no. L29324; CTR region, accession no. AF295925; and RT region, accession no. L29323) and the corresponding modules in the other Tn5252-like elements are represented as gray boxes. (A) Reference structure of the Tn5253 prototype, arbitrarily represented since only partial sequence data are available, based on references 2, 14, 15, 18, and 19. (B) Composite element (Tn5253-like) from isolate Pn1, largely matching the Tn5253 prototype. The Tn916 family transposon is a typical Tn916, with an opposite direction and a different insertion site into Tn5252 compared to the Tn5253 prototype. (C) Composite element (Tn6058) from isolate Pn20, harboring Tn6002 (with the same orientation and insertion site into Tn5252 as Tn916 in isolate Pn1) as the Tn916 family transposon. (D) Composite element (Tn1311) from isolate Pn6, harboring the SpnRi3erm(B)-like element as the Tn916 family element, inserted into a Tn5252-like element with a deleted left terminal region.

The composite element from isolate Pn1 largely matched Tn5253 in size, organization, and sequence, although Tn916 had an opposite direction and its insertion site (at nucleotide 234 of the Tn5252 CTR region; accession no. AF295925) was different from that of Tn5251 in Tn5252 (19) (accession no. X90940 and X90941).

The composite element from isolate Pn20 displayed a new organization, with Tn6002 as the Tn916 family transposon inserted in Tn5252. The resulting Tn5253-related element (67,819 bp; accession no. FM201786) was designated Tn6058 (Fig. 1C). Identities to the three Tn5252 prototype regions were high: LT, 97.0%; CTR, 97.8%; and RT, 99.3%. In Tn6058, Tn6002 had the same orientation and the same insertion site in Tn5252 as Tn916 in isolate Pn1. Both insertion site and orientation were confirmed to be the same in the other four isolates sharing Tn6058. A large portion (almost 16 kb) of Tn6058, formed largely by cargo genes and including the pC194 plasmid, displayed high identity (97.3%) to a corresponding sequence of the Tn5253-related transposon Tn2008 (11) found in the sequenced genome of S. pneumoniae CGSP14, a virulent and multidrug-resistant invasive strain (accession no. CP001033). However, unlike Tn6058, Tn2008 carries the integrase gene int_Sp23FST81.

A new organization was also displayed by the composite element from isolate Pn6, which carried the SpnRi3erm(B)-like element as the Tn916 family element. The resulting Tn5253-related element (75,303 bp; accession no. FN667862) was designated Tn1311 (Fig. 1D). The SpnRi3erm(B)-like element was flanked by sequences (left, 583 bp; right, 1,595 bp) corresponding to an ORF encoding a metallopeptidase (72.5% identity) found in the genome of Streptococcus equi subsp. equi (accession no. FM204883), suggesting a possible origin of the element from this species. The SpnRi3erm(B)-like element, flanked by these S. equi sequences, was inserted into the Tn5252-like element of Tn1311 in the area between the pC194 plasmid and the CTR region, approximately 12 kb from the latter (at nucleotide 30398). The LT region of the Tn5252-like element of Tn1311 lacked the UV resistance gene cluster of the prototype sequence (accession no. L29324). The Tn1311 backbone displayed high identity (>99%) to the very recently sequenced genome of S. pneumoniae 670-6B (accession no. CP002176).

Characterization of Tn5253-like composite elements positive for int_Sp23FST81.

In the 13 isolates positive for int_Sp23FST81, PCR experiments with suitable primer pairs were performed to understand the structure of the Tn5252-like element, seek a linkage with the associated Tn916-like transposon, and elucidate the genome integration site. The results indicated that in all 13 isolates the Tn916-like element was inserted into a Tn5252-like element forming various Tn5253-like composite structures (Table 1), all sharing the specific site of ICESp23FST81 integration into the pneumococcal genome, flanked by a 16-bp tandem duplication, near the 3′ end of gene rplL (8) (accession no. FM211187).

Eleven of the 13 isolates displayed an organization of the composite element closely resembling that of ICESp23FST81 (8) (Fig. 2A and B). The associated Tn916 family element varied (four isolates had Tn916, three had Tn6002, three had Tn2009, and one had Tn3872), with the overall size of the Tn5253-like element varying accordingly, but its insertion site was always the same, in the area between the CTR and the RT regions.

FIG. 2.

Genetic organization and available sequences of the Tn5253-like pneumococcal elements positive for int_Sp23FST81. A black bar representing the genome denotes sequencing analysis, a white bar indicates just amplification analysis (with sequencing limited to particular areas). Integrase ORFs (int_Sp23FST81 and int₉₁₆) are depicted as black arrows. Tn916 family transposons are represented as dotted bars, with a broken outline to denote the possible occurrence of different family elements (as indicated). The modules corresponding to the LT, CTR, and RT regions are represented as gray boxes. (A) Reference structure of ICESp23FST81 (accession no. FM211187). (B) Variants of ICESp23FST81 from isolates Pn8, Pn14, Pn3, and Pn13, containing Tn916, Tn6002, Tn2009, and Tn3872, respectively, as the Tn916 family transposon. (C) ICE6094 from isolate Pn19 (accession no. FR670347). An ICE6094 variant, containing Tn3872 instead of Tn6002 as the Tn916 family transposon, was detected in isolate Pn12.

The other two isolates (Pn12 and Pn19) shared a distinctive new organization of the composite element, designated ICE6094. Based on PCR analysis, the Tn5252-like element appeared to be identical in the two isolates but substantially different from both ICESp23FST81 and the Tn5252 prototype (Fig. 2C). The only difference between the two isolates was in the Tn916-like element harbored in the ICE6094 structure: Tn3872 in Pn12 and Tn6002 in Pn19. Unlike ICESp23FST81 and the composite elements of the 11 isolates carrying int_Sp23FST81, where the Tn916-like element is inserted between the CTR and the RT regions, in ICE6094 the Tn916-like element is inserted in the LT region, between int_Sp23FST81 and the mobilization genes, and has an opposite orientation. In particular, in ICE6094 from isolate Pn19, whose Tn5252-like backbone was completely sequenced (61,030 bp; accession no. FR670347), the Tn6002 insertion site was found at nucleotide 10910 of the sequence. An ∼13.5-kb area to the right of Tn6002 displayed high identity to corresponding areas of Tn1311 (97.3%) and the genome of S. pneumoniae G54 (99.6%), another multidrug-resistant clinical isolate whose genome has been completely sequenced (accession no. CP001015).

Transferability of Tn5253-like composite elements.

Nine isolates, three carrying composite elements positive for int₅₂₅₂ and six carrying composite elements positive for int_Sp23FST81, were tested as donors in mating experiments. The former included Pn1 (harboring a Tn5253-like element), Pn20 (harboring Tn6058), and Pn6 (harboring Tn1311). The latter included Pn3, Pn8, Pn13, and Pn14 (harboring ICESp23FST81 variants containing Tn2009, Tn916, Tn3872, and Tn6002, respectively, as the Tn916 family transposons) and Pn12 and Pn19 (harboring ICE6094 containing Tn3872 and Tn6002, respectively, as the Tn916 family transposon). The results are summarized in Table 2.

TABLE 2.

Conjugal transfer of resistance determinants from S. pneumoniae donors with different genotypic characteristics to the susceptible recipient S. pyogenes 12RF^a

Donor			Selection for resistance	Transfer frequency	Transconjugants
Isolate	Composite element (Tn916 family transposon)	Genotype			Genotype	MIC (μg ml−1)
						CHL	TET	ERY
Pn1	Tn5253-like (Tn916)	catpC194 tet(M)	CHL	1.6 × 10−7	catpC194 tet(M)	>32	64
Pn3	ICESp23FST81-like (Tn2009)	catpC194 tet(M) mef(E)	CHL	2.8 × 10−8	catpC194 tet(M) mef(E)	32	64	16
			ERY	5.7 × 10−8	catpC194 tet(M) mef(E)	32	64	16
Pn6	Tn1311 (SpnRi3erm(B)-like)	catpC194 erm(B) aphA-3	CHL	NDTb
			ERY	NDT
Pn8	ICESp23FST81 (Tn916)	catpC194 tet(M)	CHL	3.4 × 10−5	catpC194 tet(M)	32	64
			TET	3.7 × 10−7	catpC194 tet(M)	>32	128
Pn12	ICE6094 (Tn3872)	catpC194 tet(M) erm(B)	CHL	8.0 × 10−9	catpC194 tet(M) erm(B)	>32	64	>128
			ERY	NDT
Pn13	ICESp23FST81-like (Tn3872)	catpC194 tet(M) erm(B)	CHL	1.9 × 10−8	catpC194 tet(M) erm(B)	>32	64	>128
			ERY	NDT
Pn14	ICESp23FST81-like (Tn6002)	catpC194 tet(M) erm(B)	CHL	3.4 × 10−6	catpC194 tet(M) erm(B)	32	64	>128
			ERY	2.2 × 10−8	catpC194 tet(M) erm(B)	32	64	>128
Pn19	ICE6094 (Tn6002)	catpC194 tet(M) erm(B)	CHL	6.1 × 10−6	catpC194 tet(M) erm(B)	>32	128	>128
			ERY	1.5 × 10−6	catpC194 tet(M) erm(B)	>32	128	>128
Pn20	Tn6058 (Tn6002)	catpC194 tet(M) erm(B)	CHL	3.0 × 10−7	catpC194 tet(M) erm(B)	>32	128	>128
			ERY	2.4 × 10−7	catpC194 tet(M) erm(B)	>32	128	>128

^aNo detectable transfer was obtained when S. pneumoniae R6RF was used as the recipient.

^bNDT, no detectable transfer.

No detectable transfer was obtained from any donor when S. pneumoniae R6RF was used as the recipient. Detectable transfer from eight of the nine donors was obtained, at variable frequencies (10⁻⁵ to 10⁻⁹), with S. pyogenes 12RF as the recipient. All transconjugants exhibited the expected CHL, TET, and/or ERY resistances, even though the MIC levels expressed in the S. pyogenes host were often higher than those expressed in the pneumococcal donors. Transconjugants were obtained from the two donors harboring composite ICEs with Tn3872 as the associated Tn916 family transposon (Pn12 and Pn13) only when CHL was used for the selection. Pn6, harboring the SpnRi3erm(B)-like element, i.e., a deleted form of previously described SpnRi3erm(B), which is also nonmobile (6), was the sole donor from which transconjugants could not be obtained.

Concluding remarks.

All 20 clinical isolates of S. pneumoniae, selected on the basis of cat_pC194-mediated CHL resistance, proved to harbor a Tn5253-like element. These composite elements demonstrated substantial heterogeneity, consistent with the belief that element recombination is frequent and that module swapping can result in the emergence of new elements (33). The only correlation between typing data and particular composite elements was the fact that four of the five isolates harboring Tn6058 belonged to serotype 3 and PFGE type A. Such heterogeneity confirms and affords further insights into the diversity of putative Tn5253-like pneumococcal elements, which has recently been reported by Henderson-Begg et al. (14) on the basis of combined analysis of selected genes and antibiogram data. Remarkably, that study also underscored that these composite elements appear to be prevalent among internationally recognized pandemic clones of S. pneumoniae. The present findings clarify that the overall heterogeneity of Tn5253-like pneumococcal elements results from at least two levels of variability: in the Tn5252-like and in the Tn916-like transposons.

At the first level, at least two variants of the Tn5252-related transposon can be distinguished to begin with, depending on the presence of the int₅₂₅₂ or the int_Sp23FST81 gene. The two integrase genes drive the integration of their elements into the pneumococcal genome at specific sites, i.e., into the spr1043 gene (R6 genome) and near the 3′ end of gene rplL, respectively. Moreover the Tn5252-like elements show further variability in their genetic organization and structure regardless of the int gene. While the CTR and RT regions seem to be steadier modules, the LT region, aside from the int gene, seems to be more changeable (e.g., it lacks the UV resistance gene cluster in Tn1311 and is split by the insertion of the Tn916-like element between the integration and the mobilization genes in ICE6094). Furthermore, a variety of different cargo genes make the intermodular areas largely unpredictable.

A second level of variability is related to the Tn916 family transposon, which is inserted (at variable sites, consistent with the poor specificity of int₉₁₆ [23, 33]) into the Tn5252-like transposon. The Tn916-like transposon may be Tn916 or another element from a number of Tn916 family pneumococcal relatives carrying a variety of insertions containing erythromycin resistance genes such as erm(B) or mef(E) in addition to the tet(M) tetracycline resistance determinant typical of the family (22, 29). It is noteworthy that Tn916 family pneumococcal transposons such as Tn1545 and Tn6003, containing a complete MAS fragment with a second erm(B) gene lacking the stop codon (5), were not found in Tn5253-like elements in this study.

While Tn916 family transposons can be found both as independent elements and inserted into Tn5252-related transposons to form a Tn5253-like composite element, Tn5252-related transposons, whether carrying int₅₂₅₂ or int_Sp23FST81, are not known to occur naturally outside a composite element, i.e., as independent genetic elements. It is worth noting that S. pneumoniae SP1000, a historical strain used in early studies of the Tn5252 transposon (15, 18, 24, 26, 27, 30), was a laboratory-derived organism obtained by deleting Tn5251 from Tn5253 (2).

Our failure to obtain detectable transfer in mating experiments using S. pneumoniae R6RF as the recipient probably reflects the poor propensity of pneumococci to acquire foreign genetic material via conjugation. In contrast, conjugal transfer was mostly successful when S. pyogenes 12RF was used as the recipient. The sole nonmobile composite element (Tn1311, isolate Pn6) contains a Tn916-related element, the SpnRi3erm(B)-like element, which, despite the occurrence of int₉₁₆ and xis₉₁₆, appears to be a less typical member of the Tn916 family. In some instances, being part of a composite element may give some Tn916 family transposons, which as independent genetic elements are apparently nontransferable in conjugation experiments, an opportunity to be mobilized. This might be the case for Tn2009, which is nontransferable by conjugation in both inter- and intraspecific mating assays (9, 10), and for Tn3872, whose nontransferability by conjugation, despite exceptions with streptococcal donors other than pneumococci, is extensively documented (16, 29). Remarkably, both composite Tn5253-like elements bearing Tn3872 as the Tn916-like transposon (ICE6094 in isolate Pn12 and the ICESp23FST81-like element in isolate Pn13) turned out to be transferable only when selection was for CHL resistance, whose determinant is located on the Tn5252-like transposon. TET or ERY (clearly not CHL) was used for selection in previous studies reporting nontransferability of Tn3872.