Different Genetic Supports for the tet(S) Gene in Enterococci

Abstract

The diversity of tet(S) genetic contexts of 13 enterococci from human, animal, and environmental samples from different geographical areas is reported. The tet(S) gene was linked to either CTn6000 variants of chromosomal location or composite platforms flanked by IS1216 located on plasmids (∼40 to 115 kb). The comparative analysis of all tet(S) genetic elements available in the GenBank databases suggests that CTn6000 might be the origin of a variety of tet(S)-carrying platforms that were mobilized to different plasmids.

TEXT

The tetracycline resistome comprises over 1,189 genes identified in more than 84 Gram-positive and 354 Gram-negative species (17), which included 43 fully characterized tet genes coding for resistance to tetracycline by different mechanisms (http://faculty.washington.edu/marilynr). Genes involved in ribosomal protection, such as tet(S), tet(M), tet(O), tet(Q), and tet(W), were identified in conjugative transposons (CTns), phage-derived structures, and/or conjugative plasmids (3, 4, 5, 11, 12, 16). The tet(S) gene has been found among Firmicutes and gammaproteobacteria from diverse ecological sources since the early 1950s (1, 2, 10, 11) (http://faculty.washington.edu/marilynr/tetweb2.pdf), and several genetic platforms carrying tet(S), such as CTn6000, CTn916S, pKL0018, and pK214, have been described (5, 9, 11). Among enterococci, the genetic context of tet(S) has been characterized in only a single Enterococcus casseliflavus isolate from animal origin (5, 15). In this work, we described the genetic platforms of 13 tet(S)-carrying enterococci from different origins and geographical areas and compared them with sequences available in GenBank databases. This study gives for the first time a snapshot of the epidemiology and dynamics of the tet(S) gene by describing its location in different CTn6000 variants differing in insertions and deletions as well as in composite genetic platforms flanked by insertion sequences. Such diversity of genetic platforms seems to have facilitated its spread among diverse clonal backgrounds by different recombinatorial processes.

Antibiotic susceptibility testing was performed by standard methods (6), and characterization of other genes coding for resistance to tetracycline [tet(M), tet(L), tet(K), and tet(O)] was accomplished by PCR (1). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) (http://www.mlst.net) (14). The 13 enterococci carrying tet(S) were identified as Enterococcus faecalis (n = 5), Enterococcus faecium (n = 3), Enterococcus gallinarum (n = 2), and other Enterococcus species (n = 3). Epidemiological details of the strains studied appear in Table 1. Isolates expressed resistance to tetracycline (100%; n = 13) and minocycline (92%; n = 12) and frequently also harbored tet(M) (54%; n = 7) or tet(L) (31%; n = 4).

Table 1.

Epidemiological and genetic background of enterococci carrying tet(S)^d

Species	PFGE type	MLST clonal complex (sequence type)	Yr	Source	Sample	No. of isolates	Geographical location	Antibiotic resistance phenotype	Antibiotic resistance genotype	tet(S) location (size)	Genetic context of tet(S)
E. casseliflavusa	ND		1993	Monkey	Feces	1	Alberta, Canada	TET, STR	tet(S)	Chr	CTn6000 type A
E. faecalis	HV70	CC55 (ST55)	2001	Healthy human	Feces	1	North Portugal	TET, MIN, ERY, CHL, STR	tet(S), tet(M), erm(B)	Chr	CTn6000 type C
	I	(ST158)	2002	Hospitalized human	Wound, blood	1	Michigan, USA	TET, MIN, ERY, CIP, GEN, STR, VAN, TEC	tet(S), erm(B), vanA	Chr	ΔCTn6000 type D
	H	CC87 (ST28)	2002	Hospitalized human	Unknown	1	Belgrade, Serbia	TET, MIN, ERY, CIP, CHL, STR	tet(S), tet(M), erm(B)	PL (50 kb)	tet(S) platform type G
	SN242	(ST445)	2006	Piggery Cb	Manure, residual water	2	Central Portugal	TET, MIN, ERY, CIP, CHL, STR	tet(S), tet(M), tet(L), erm(B)	PL (115 kb)	tet(S) platform type H or I
E. faecium	A71	CC22 (ST32)	2008	Fountain	Drinking water	1	North Portugal	TET, MIN, ERY, CIP	tet(S)	Chr	CTn6000 type B
	SN210	CC22 (ST32)	2006	Piggery Cb	Antisepticc	1	Central Portugal	TET, MIN, ERY, CIP	tet(S), tet(M), tet(L)	Chr	CTn6000 type C
	Z	CC17 (ST17)	2002	Hospital sewage	Water	1	North Portugal	TET, MIN, ERY, CIP, GEN, STR, AMP	tet(S), tet(M), erm(B)	PL (40 kb)	tet(S) platform type G
E. gallinarum	F60		1999	Poultry	Carcass	1	North Portugal	TET, ERY, CIP, STR, VAN	tet(S), tet(M), erm(B), vanC1	PL (70 kb)	tet(S) platform type E
	F61		1999	Poultry	Carcass	1	North Portugal	TET, MIN, ERY, VAN	tet(S), tet(L), erm(B), vanC1	PL (45 kb)	tet(S) platform type E
Enterococcus spp.	SN244		2006	Piggery Ab	Waste lagoon	1	South Portugal	TET, MIN	tet(S)	Chr	CTn6000 type C
	SN243		2006	Piggery Cb	Food for animals	1	Central Portugal	TET, MIN, ERY, CIP, CHL, STR	tet(S), erm(B)	PL (115 kb)	tet(S) platform type H
	SN245		2006	Piggery Bb	Waste lagoon	1	South Portugal	TET, MIN, ERY, CIP	tet(S), erm(B)	PL (70 kb)	tet(S) platform type F

^aThis strain was previously characterized by Brouwer et al. (5), but it was included in Table 1 for comparison with enterococci carrying CTn6000 that were analyzed in this study.

^bDifferent piggeries are identified with letters A, B, and C.

^cAntiseptic solution used in piglets' skin after birth.

^dAbbreviations: TET, tetracycline; MIN, minocycline; ERY, erythromycin; CIP, ciprofloxacin; CHL, chloramphenicol; STR, high level of resistance (HLR) to streptomycin; GEN, HLR to gentamicin; VAN, vancomycin; AMP, ampicillin; ND, not determined; Chr, chromosome; PL, plasmid.

Hybridization of I-CeuI/S1-digested genomic DNA with tet(S) and 23S rRNA probes demonstrated either a chromosomal (n = 5) or plasmid (n = 8; ∼40 to 115 kb) location of the tet(S) gene (Table 1) (8). Screening of CTns included the identification of integrases and excisionases of different CTns previously found to carry tet(S) as CTn6000 or CTn916 and further characterization of the transposon's backbone by PCR mapping based on available sequences (Table 2 and Fig. 1) (13). The tet(S) gene was located at a chromosome within platforms carrying int/xis_CTn6000 sequences (n = 5) or on plasmids within platforms lacking int/xis sequences of any CTn tested (n = 8) (Fig. 1 and Table 1). The different tet(S) platforms were designated by capital letters. Characterization of chromosomal CTn6000 platforms was established by comparison of restriction fragment length polymorphism (RFLP) patterns obtained after digestion of PCR products with TaqI, AluI, DdeI, HindIII, or EcoRI and by the DNA sequences of representative fragment types (Fig. 1). Three variants, arbitrarily named types “B,” “C,” and “D” to differentiate them from the original CTn6000 (5), designated type “A” here, were identified (Table 1 and Fig. 1). Type C (GenBank accession no. JN208881) was found in Portugal in an ST55 (CC55) E. faecalis isolate identified in five different healthy humans and in one ST32/CC22 E. faecium strain and one Enterococcus species strain isolated from two different piggeries. Type C differs from type A by the lack of the 1,830-bp insertion within orf14 consisting of a group II intron and an additional 17 bp. Type B (GenBank accession no. JN208880), isolated from an ST32/CC22 E. faecium strain from a fountain's water in Portugal, contains an ISEfm2 insertion with identifiable boundaries within the orf13-tet(S) region. Finally, type D, carried by an ST158 E. faecalis clone from Michigan, was able to be only partially identified. It carried int_CTn6000-tet(S) and the orf23-orf29_CTn6000 region, although they were not able to be directly linked by using different PCR overlapping assays between different CTn6000 regions. Comparative analysis of the CTn6000 elements described in this study with sequences available at the GenBank database resulted in the detection of three unpublished genetic elements identical to CTn6000 variant C within the full-genome sequences of three clinical American isolates, two E. casseliflavus strains and an ST9/CC9 E. faecalis β-lactamase-producing strain causing a large outbreak in the United States in 1986 (GenBank accession numbers NZ_GG692817.1, NZ_GG670387.1, and NZ_AEBZ01000002.1) (http://www.broadinstitute.org/annotation/genome/enterococcus_faecalis/GenomeDescriptions.html). Conversely, variants A and B were not detected in the GenBank databases. The analysis of the chromosomal integration sites of CTn6000 variants was performed as described previously (15) and revealed that types B and C use the gene coding for the L31 ribosomal protein as the hot spot for integration, as reported for the original CTn6000 type A (15). However, the L31-encoding gene was not the integration site for type D.

Table 2.

Strategies and conditions used for characterizing CTn6000

Fig 1.

Diversity of complete and partial CTn6000 backbones and other genetic platforms carrying tet(S). Boxes in gray indicate genes absent in CTn6000. Sequencing was accomplished for CTn6000 types C (fully), B, and D (partially). Amplicons of CTn6000 types B and D with RFLP patterns common to types A and C were not sequenced. *, this strain also carried the orf23-orf29 CTn6000 region, although it was not able to be directly linked to the int_CTn6000-tet(S). The boundaries of tet(S) were sequenced in tet(S) platform types E, F, G, H, and I. Dotted lines in IS1216 and tet(S) gene schemes indicate that they were partially sequenced. **, the tet(S) gene is annotated as tet(M) in the sequence in GenBank under accession number GQ900487.1.

The tet(S) platforms located on plasmids corresponded to three E. faecalis isolates (2 PFGE types), one E. faecium isolate, two E. gallinarum isolates, and two Enterococcus species isolates obtained from different hosts and arbitrarily designated types “E,” “F,” “G,” “H,” and “I” (Table 1 and Fig. 1). They contained IS1216 copies targeting different positions within orf13_CTn6000 (types E and F), the orf13_CTn6000-tet(S) intergenic region (types G and H), and downstream orf6_CTn6000 (type F). Type I comprises a copy of ISEnta1 previously described in an E. faecalis isolate (AY884205.1) and a partial sequence of cadE, a gene linked to cadmium resistance which was also identified in type H. All sequences corresponding to orf13, the orf13-tet(S) intergenic region, and orf6 showed a high degree of identity (≥99%) with the original CTn6000 sequence.

Conjugative transfer of the CTn6000 variants A, B, C, and D was tested as previously described (15). Wild-type and recipient strains (E. faecalis JH2-2, E. faecium BM4105RF, and Staphylococcus aureus 8325) were used in a ratio of 1:1. Transconjugants were selected in BHI agar supplemented with tetracycline (10 mg/liter), rifampin (25 mg/liter), and fusidic acid (5 mg/liter) and confirmed by resistance to the three antibiotics (disk diffusion) and by PCR amplification of CTn6000 int/xis and/or tet(S). We did not find any isolate fulfilling all these features, possibly due to the low conjugation frequency (10⁻⁹) of CTn6000 (15). Transfer of plasmids carrying tet(S) within each type of platform characterized was also not achieved.

This study documents the spread of tet(S) among enterococcal species from different origins mediated by CTn6000 or other genetic platforms formed by composite structures often located on plasmids. Comparative analysis of the sequences corresponding to all tet(S) elements available at GenBank to date reflects the dynamics of tet(S) platforms by hosting different transposable elements involved in further arrangements mediated by insertion elements, often resulting in IS1216 composite platforms.

Further analysis of enterococcal genomes and extrachromosomal genetic elements is necessary to fully understand the evolutionary “transferability” of tet(S) genetic contexts, that is, the ability to be successfully transferred and retained in different replicons and the resulting impact on the diversification and evolution of bacterial population structure (7).