Characterization of Tn5801.Sag, a Variant of Staphylococcus aureus Tn916 Family Transposon Tn5801 That Is Widespread in Clinical Isolates of Streptococcus agalactiae

Abstract

Tn5801, originally detected in Staphylococcus aureus Mu50, is a Tn916 family element in which a unique int gene (int₅₈₀₁) replaces the int and xis genes in Tn916 (int₉₁₆ and xis₉₁₆). Among 62 tet(M)-positive tetracycline-resistant Streptococcus agalactiae isolates, 43 harbored Tn916, whereas 19 harbored a Tn5801-like element (Tn5801.Sag, ∼20.6 kb). Tn5801.Sag was characterized (PCR mapping, partial sequencing, and chromosomal integration) and compared to other Tn5801-like elements. Similar to Tn5801 from S. aureus Mu50, tested in parallel, Tn5801.Sag was unable to undergo circularization and conjugal transfer.

TEXT

Tn916 family elements (1, 2) are broad-host-range elements, widespread in Gram-positive bacteria, that mostly exhibit the distinctive properties of integrative and conjugative elements (ICEs) (3). Their open reading frames (ORFs) are organized into functional modules (conjugation, recombination, transcriptional regulation, and accessory functions): albeit with well-known variations (1, 2), the recombination module mostly consists of an integrase (int₉₁₆) gene and an excisionase (xis₉₁₆) gene, and the accessory gene is typically the tetracycline (TET) resistance determinant tet(M).

The best-known Tn916 family element from Staphylococcus aureus is Tn5801 (∼25.8 kb) (2), which was detected in the genome of Mu50 (DDBJ accession no. BA000017) (4), a well-established methicillin-resistant and vancomycin-intermediate Japanese clinical isolate (5). Tn5801, regarded as one of the nine genomic islands in the Mu50 genome (6), shows a modular organization similar to that of Tn916 and has several similar ORFs. However, besides the presence of additional ORFs, whose functions are largely unknown, DNA identities are rather low except in the case of tet(M) (97.7%). In particular, the recombination module differs from that of Tn916, as it lacks the xis gene and shows very low DNA identity (38.6%) between int₅₈₀₁ and int₉₁₆. This organization closely resembles that found in CW459tet(M), a genetic element from Clostridium perfringens CW459 (GenBank accession no. AF329848) (7).

Tn5801-like transposons have been detected in other human isolates of S. aureus (8, 9); in one case the element, Tn6014 from S. aureus 1680, was able to transfer, at low frequency, to S. aureus recipients (8). Among streptococci, a Tn5801-like element has been described for Streptococcus mitis B6 (EMBL accession no. FN568063) (10).

In the present study, we showed that a Tn5801-like transposon, designated Tn5801.Sag, is found in about 30% of TET-resistant clinical isolates of Streptococcus agalactiae, a species in which TET resistance is around 90% worldwide. The genetic organization of Tn5801.Sag was determined and compared with that of other Tn5801-like elements, and the putative core site was identified. Similar to Tn5801 from S. aureus Mu50, which was tested in parallel in this study, Tn5801.Sag was unable to undergo circularization and conjugal transfer.

All PCR primers used are shown in Table 1.

Table 1.

Oligonucleotide primer pairs used

Procedure and gene/amplicon	Primer designation	Sequence (5′–3′)	Reference or source	Product size (bp)
Detection of TET resistance genes
tet(M)	TETM3	ATGGAAGCCCAGAAAGGAT	11	740
	TETM2	GAACTCGAACAAGAGGAAAGC	11
tet(O)	TETO1	AACTTAGGCATTCTGGCTCAC	11	519
	TETO2	TCCCACTGTTCCATATCGTCA	11
PCR evidence of Tn916
int916	int-for	GCGTGATTGTATCTCACT	12	1,046
	int-rev	GACGCTCCTGTTGCTTCT	12
xis916	xis-for	AAGCAGACTGAGATTCCTA	13	194
	xis-rev	GCGTCCAATGTATCTATAA	13
orf7-orf8a	O15	GTACGTCCACCAATGTGG	14	902
	O16	GCACGCTTCCACGAAAGGAG	14
orf20-IR18–19a	J12	CCCATTGAAGACGCAGAAGT	15	801
	J11	AAAAATCCCTACCGCACT	15
orf24-orf20a	TN6-rev	CCATCAAACATTCATTCAGC	15	3,358
	J13	GGTTTTGTGGTTAGTTTT	15
PCR mapping of Tn5801.Sagb
int5801	1812	GTCCATACGTTCCTAAAGTCGTC	8	726
	1811	CCGATATTGAGCCTATTGATGTG	8
sav400	400R	TCGTATTTCAAGGCTTCGTC	This study	369
	400F	TACCGAAGAGTCCATCAAAC	This study
sav408	408R	AATGTAGGGGCGACTTGATG	This study	1,005
	408F	ACTGGCTTATGGCGTTTCTC	This study
sav409	409R	GCAGACAAACCAAGATAAGC	This study	940
	409F	GAGAGCGAATCAAAGCCAAC	This study
sav413	413R	AACACCGTTGTCGTCTCCAC	This study	743
	413F	TTGCTAGTAATATAAGGGCGA	This study
sav414	414R	ATTAGATACACAACATCCTCATC	This study	579
	414F	ACAGGCAATCCCATCAGAAC	This study
sav415	415R	TAGATGAGGCTTGATACACC	This study	677
	415F	TTCTCGTAACGGCTCCTATG	This study
int5801-tet(M)	1812	GTCCATACGTTCCTAAAGTCGTC	8	4,971
	TETM2	GAACTCGAACAAGAGGAAAGC	11
tet(M)-sav400	TETM3	ATGGAAGCCCAGAAAGGAT	11	3,362
	400F	TACCGAAGAGTCCATCAAAC	This study
sav400-sav408	400R	TCGTATTTCAAGGCTTCGTC	This study	9,331
	408F	ACTGGCTTATGGCGTTTCTC	This study
sav408-sav409	408R	AATGTAGGGGCGACTTGATG	This study	2,612
	409F	GAGAGCGAATCAAAGCCAAC	This study
sav409-sav411	409R	GCAGACAAACCAAGATAAGC	This study	2,142
	411F	GAGATTAGCAGAAGGTATTGTG	This study
sav409-sav413	409R	GCAGACAAACCAAGATAAGC	This study	3,874
	413F	TTGCTAGTAATATAAGGGCGA	This study
sav413-sav414	413R	AACACCGTTGTCGTCTCCAC	This study	1,968
	414F	ACAGGCAATCCCATCAGAAC	This study
sav414-sav415	414R	ATTAGATACACAACATCCTCATC	This study	3,295
	415F	TTCTCGTAACGGCTCCTATG	This study
Tn5801.Sag chromosomal integration sitec
SAG967 (guaA)	LJ967	CGTGAAGAAATCGCTAAAG	This study	1,228
int5801	1811	CCGATATTGAGCCTATTGATGTG	8
sav411	CF1	TTCAAAGGAACAGAAGCGGG	This study	1,414
SAG964	RJ964	GAAGTAGAAGAGAGCCATAG	This study
Search for circular form
int5801	1811	CCGATATTGAGCCTATTGATGTG	8
sav411	CF1	TTCAAAGGAACAGAAGCGGG	This study

^aORFs numbered according to the reported organization of Tn916 (GenBank accession no. U09422).

^bTn5801 from the genome of S. aureus Mu50 (DDBJ accession no. BA000017; sav genes) was used as the reference sequence. In PCR assays, S. aureus Mu50 (ATCC 700699) was used as a positive control and S. pneumoniae BM4200 (Pasteur Institute Collection), harboring the Tn916-like transposon Tn1545 (1), was used as a negative control.

^cThe genome of S. agalactiae strain 2603V/R (GenBank accession no. AE009948; SAG genes) was used as the reference sequence.

Characterization of TET-resistant S. agalactiae isolates.

Sixty-nine clinical isolates of S. agalactiae, recovered in laboratories of central Italy in 2010–2011 and confirmed as being Lancefield group B using Slidex Strepto Plus (bioMérieux, Marcy l'Étoile, France), were used. Of them, 64 (93%) were TET resistant (MICs, ≥8 μg/ml). PCR assays demonstrated that tet(M) and tet(O) were the sole tet genes in 58 and 2 isolates, respectively; 4 isolates carried both determinants. Among the 62 tet(M)-positive isolates, 43 yielded positive PCRs for int₉₁₆ and xis₉₁₆; the remaining 19 were negative for both genes as well as for three additional regions of the transposon. However, sequence analysis of the tet(M) amplicon, performed in 3/19 randomly selected isolates, showed 100% DNA identity to the corresponding tet(M) portion of Tn5801 from S. aureus Mu50 (4). This finding prompted us to look for int₅₈₀₁, the integrase gene of Tn5801, which was found in all 19 isolates. The latter fell into several serotypes and pulsotypes (data not shown), thus excluding that they represented a clonal population.

Characterization and comparative analysis of Tn5801 from S. agalactiae (Tn5801.Sag).

The 19 int₅₈₀₁-positive isolates underwent PCR mapping using the primers and strategies summarized in Table 1 and Fig. 1A. All isolates yielded comparable results, with positive PCRs and amplicons of the expected sizes obtained with all but six of the relevant primer pairs. Specifically, negative reactions were obtained with those pairs in which at least one primer targeted one of the last three ORFs of Tn5801 from S. aureus Mu50 (sav413, sav414, and sav415), which thus appeared not to be found in Tn5801 from S. agalactiae (designated Tn5801.Sag).

Fig 1.

Schematic representation of Tn5801.Sag from S. agalactiae strain 14774 (A) and its chromosomal integration (left and right junctions) (B). (A) Tn5801.Sag was determined by PCR mapping and sequencing of two regions. The primers used are listed in Table 1. The mapping strategy is outlined in the upper portion (the amplicons used to detect individual ORFs, i.e., obtained by pairing two primers internal to the same ORF, are not shown). The two regions sequenced initially (7,593 bp, left, and 3,814 bp, right) are indicated by horizontal bars. Tn5801.Sag is compared to Tn5801 from S. aureus Mu50, where ORFs are numbered sav392 to sav415 according to the original designations (DDBJ accession no. BA000017); percent DNA identities are reported in gray areas between sequenced regions. tet(M) and int₅₈₀₁ are represented as black and spotted arrows, respectively. (B) Tn5801.Sag was integrated at the 3′ end of the guaA gene. This gene, detected in all S. agalactiae genomes sequenced to date, corresponds to ORF967 from S. agalactiae 2603V/R (GenBank accession no. AE009948), from which chromosomal ORF designations derive. The amplicons obtained by pairing primers LJ967/1811 (left junction) and CF1/RJ964 (right junction), whose sequencing extended the two portions of Tn5801.Sag sequenced initially, are shown as bars. tet(M) and int₅₈₀₁ are represented as black and spotted arrows, respectively, and other Tn5801.Sag ORFs as white arrows; chromosomal ORFs are depicted as gray arrows.

One of the 19 isolates (strain 14774) was used in DNA sequencing experiments, performed as described elsewhere (16). Two amplicons, yielded by primer pairs 1812/400F [7,593 bp, encompassing the tet(M) gene and most int₅₈₀₁] and 408R/411F (3,814 bp), were sequenced (EMBL accession no. HF930766). The two sequenced regions of Tn5801.Sag displayed 97.1% [tet(M), 100%] and 96.5% DNA identities with the corresponding regions of Tn5801 from S. aureus Mu50 (Fig. 1A) and 96.9% [tet(M), 99.5%] and 96.5% with those of S. mitis B6; the former sequence displayed 96.1% identity [tet(M), 100%] with the corresponding region (the only one that has been sequenced; GenBank accession no. EU918655) of Tn6014 from S. aureus 1680. Greater identities [99.9% and 100%; tet(M), 100%] were recorded with the corresponding regions of E. faecalis 62, for which a “Tn916 element” (not identified as Tn5801-like) was reported (17) in the sequenced genome (GenBank accession no. CP002491). The latter element (∼20.6 kb) was very similar to Tn5801.Sag (∼20.6 kb based on sequencing and PCR mapping data) also as to ORF organization: in particular, the two elements share the lack of the last three ORFs of Tn5801 from S. aureus Mu50 (sav413, sav414, and sav415), located after the conjugation module and not present in Tn916. It is worth noting that the last ORF (sav415, a transposase gene) is also missing in the Tn5801-like element from S. mitis B6, in which sav413 and sav414 are present.

Chromosomal integration of Tn5801.Sag and identification of the putative core site.

The early study of CW459tet(M) (7) and later studies of genetic elements related to Tn5801 (10, 18, 19) concur in describing an integration site just downstream of guaA, a chromosomal gene encoding a GMP synthase that is consistently found adjacent to int₅₈₀₁ in the sequenced genomes containing a Tn5801 element. Using strategies refined in previous studies (16, 20–22), this site was thus explored in the 19 S. agalactiae isolates harboring Tn5801.Sag. The genome of S. agalactiae strain 2603V/R (GenBank accession no. AE009948) (23) was used as the reference sequence. As illustrated in Fig. 1B, pairing of primers LJ967/1811 (left junction) gave an ∼1.2-kb amplicon from all 19 S. agalactiae isolates; by pairing primers CF1/RJ964 (right junction), an ∼1.4-kb amplicon was obtained from all but one isolate, which yielded an ∼1-kb-larger amplicon; this was subsequently shown to reflect the presence of SAG965 and SAG966, encoding insertion sequences in the S. agalactiae 2603V/R genome that were not found in the other 18 isolates. By analyzing and comparing the two amplicon sequences from strain 14774, it was possible to determine the chromosomal junctions of Tn5801.Sag. The putative core site was an almost completely overlapping 11-bp sequence identified on the left (GAGTGGGAGTA) and right (GAGTGGGAATA) ends of the transposon; the latter sequence was identical to that found in both Tn5801 junctions of S. aureus Mu50.

Transferability studies.

Three isolates, including strain 14774, were used as donors in conjugal transfer experiments, performed as described elsewhere (24). No transconjugants were obtained with any of the three recipients used: S. agalactiae 1357RF (25), S. pyogenes 12RF (24), and S. aureus RN4220RF (26), used in the sole successful conjugative transfer of a Tn5801 element (Tn6014) reported so far (8). Similar negative results were obtained using S. aureus Mu50 (ATCC 700699) as the donor.

The apparent nontransferability of Tn5801.Sag and of Tn5801 from S. aureus Mu50 was consistent with the absence, in both cases, of an intermediate circular form, as resulting from the negative PCR obtained using the outward-directed primer pair 1811/CF1.

Conclusions.

Among the so-called Tn916-like elements (1, 2), major differences are found in the recombination module, where the prevailing two-gene organization (int₉₁₆ and xis₉₁₆) typical of Tn916 may be replaced by a single gene. This is the cases of tndX in Tn5397 from Clostridium difficile (7), a gene that in streptococci is commonly found in S. pyogenes in ICESp1116 (22); of int₄₅₉ in CW459tet(M) from C. perfringens (7); and of int₅₈₀₁ (identical to int₄₅₉) in Tn5801 from S. aureus (4). Now, the finding that in no less than 30% of TET-resistant clinical isolates of S. agalactiae—a species for which TET resistance rates are around 90%—resistance was mediated by the tet(M) gene carried by a Tn5801-like transposon (Tn5801.Sag) is a major result of this study. Accordingly, a sizable proportion (about 50/250) of S. agalactiae scaffolds and contigs currently found in GenBank harbors Tn5801.Sag. The frequent occurrence of Tn5801.Sag in S. agalactiae strengthens the notion of a composite organization of the chromosome of this species (27–29).

Subsequent to the original detection of Tn5801 in S. aureus Mu50, Tn5801-like transposons were detected in other human S. aureus isolates; one such transposon (Tn6014) was shown to be able to transfer to an S. aureus recipient (8). Conversely, Tn5801.Sag is apparently unable to transfer, like Tn5801 from S. aureus Mu50, whose actual transferability had not been tested before the present study.

As to the genetic organization of Tn5801.Sag, differences from other Tn5801-like transposons mainly involved the right terminus of the element, with the last three ORFs of Tn5801 from S. aureus Mu50 (sav413, sav414, and sav415, not present in Tn916) missing in Tn5801.Sag, while only the last one (sav415) is missing in Tn5801-like from S. mitis B6. In contrast, the left termini are very similar in all Tn5801-like transposons, and the adjacent chromosomal gene is unvaryingly guaA. Therefore, while Tn916 preferentially integrates into A·T-rich targets in a broad range of hosts (2), int₅₈₀₁ and related genes appear to code for integrases leading to site-specific recombination at the 3′ end of guaA.

Nucleotide sequence accession number.

Two new nucleotide sequences reported in this work have been deposited in the EMBL database under accession no. HF930766 .