satG, Conferring Resistance to Streptogramin A, Is Widely Distributed in Enterococcus faecium Strains but Not in Staphylococci

Abstract

A gene almost identical to satG was isolated from an Enterococcus faecium strain. This gene was transferred to a Staphylococcus aureus recipient strain where it conferred resistance to streptogramin A. satG was found to be widely distributed among E. faecium strains but not detected among staphylococci.

Streptogramin, virginiamycin, pristinamycin, and synergistin are produced by Streptomyces and consist of synergistic mixtures of two chemically different molecules: A and B compounds (10). In some European countries and in Algeria, these mixtures are used both orally and topically, mostly against staphylococcal infections. Virginiamycin is used as a growth promoter in animal feed in Europe and in the United States. Virginiamycin-resistant Enterococcus faecium is prevalent in fecal and intestinal samples from turkeys, pigs, broilers, and farmers in Europe and America (1, 14, 19, 20, 21). As bacteria can be transferred via food from animals to humans, this is alarming, in particular because quinupristin-dalfopristin (see supplement to J. Antimicrob. Chemother. volume 30 [1992]), an injectable mixture of semisynthetic streptogramins soon to be released for commercial use, is expected to be widely used, mainly to treat vancomycin-resistant E. faecium infections.

The satA gene (18) encoding an acetyltransferase that inactivates streptogramin A compounds was identified as part of a plasmid from an E. faecium isolate. It was found in only 29% of the 140 tested E. faecium strains isolated in Dutch and Danish farms and resistant to the above-mentioned mixtures (13, 14). Five of the E. faecium strains isolated in Denmark harbored a large plasmid which conferred resistance to the mixtures and which was transferable by filter mating experiments to an E. faecium recipient (13). None of the transconjugants harboring these plasmids carried satA, vat, vatB, vga, or vgaB (13). These results suggested that the E. faecium strains contained another, unidentified streptogramin A resistance gene(s). We investigated this possibility.

The 51 E. faecium strains included in this study (MICs of virginiamycin ranged from 8 to 32 mg · liter⁻¹) were those described by Jensen et al. (14). They were isolated from fecal samples from poultry (n = 22), pigs (n = 5), farmers (n = 19), and suburban residents (n = 5) in the Netherlands (Table 1). satA was previously found in 19 strains, and vgb was found in a single strain by PCR (14). The E. faecium strains were analyzed by Southern hybridization at high stringency (65°C) (2) with intragenic probes to screen for the following eight genes, previously found in staphylococcal and enterococcal plasmids conferring resistance to the mixtures: satA (18) (with primers satA1 at nucleotide [nt] positions 189 to 210 and satA2 at nt positions 760 to 782 [accession no. L12033]), vat (9), vatB (3), vatC (6), vga (7), and vgaB (4) conferring resistance to A compounds and vgb (8) and vgbB (6), encoding lactonases which hydrolyze B compounds. A total of 19 of the strains carried satA, and the combination of vat and vgb was detected in a single strain, KH6 (Table 1). vat and vgb were contiguous and in the same relative position in KH6 as in the staphylococcal plasmids in which the vat-vgb combination is carried by a DNA fragment originating from the E. faecalis plasmid pAMß1 (5).

TABLE 1.

Relevant characteristics of the 51 E. faecium strains isolated in The Netherlands

Sgr gene(s)	Size (kb) of the hybridizing HindIII fragment	Strain origin(s) (no. of isolates and city)
satA	2.3a	Turkey farmer (1)
	3.8a	Pigs (2, Weert)
	3.9	Turkey farmer (1)
	3.9a	Turkey farmer (1), chicken farmers (2), suburban inhabitants (1, Weert; 2, Roermond)
	4.0a	Broiler (1), chicken farmer (1), suburban inhabitant (1, Weert)
	4.3a	Chicken farmer (1)
	4.5a	Chicken farmer (1), pig (1, Weert)
	5.6a	Chicken farmer (1)
	6.0a	Turkey (1)
	7.0a	Broiler (1)
satG	1.4	Turkeys (3)
	1.8	Pigs (2, Weert)
	1.9	Suburban inhabitant (1, Weert)
	2.0	Chicken farmer (1), turkey farmer (1)
	2.0a	Chicken farmer (1)
	2.3	Turkey farmer (1)
	2.5	Turkey farmers (2)
	2.5a	Turkey farmer (1)
	3.6a	Broilers (3), turkey (1)
	4.3a	Broilers (5)
	5.0a	Turkeys (4), turkey farmer (1), broilers (2)
	7.3a	Turkey farmer (1)
	>10.0a	Broiler (1)
satG plus vat-vgb	4.6a	Turkey farmer (1)b
	8.9

^aThis fragment was among those detected in extrachromosomal DNA bands (≥40 kb) migrating above the chromosomal DNA fragments of the uncleaved total cellular DNA by agarose gel electrophoresis in Tris-acetate buffer. In the other strains, the hybridizing bands comigrated with the chromosomal fragments, but the hybridization signals were as strong as those of the extrachromosomal DNA, suggesting that they may be carried by plasmids. 

^bStrain KH6 was the only strain in which the vat-vgb combination was detected. 

A total of 31 of the tested E. faecium strains did not hybridize to any of the eight gene probes tested. PCR experiments were carried out at a low annealing temperature (40°C) with a pair of degenerate primers, M and N (3, 16), designed to amplify a DNA fragment from any sequence encoding a streptogramin A acetyltransferase containing two well-conserved motifs, III and IV (3, 6, 16). A DNA fragment of the expected size (147 nt) was amplified from the cellular DNA of all the strains. The amplicon obtained with strain K14 was sequenced with oligonucleotides M and N as the primers. Its sequence was only 60.4 to 68.6% similar to those of the SgA acetyltransferase genes (vat, vatB, vatC, and satA), suggesting that the amplicon was from a different gene. A 5-kb HindIII fragment hybridizing with the sequenced amplicon was isolated from the cellular DNA of strain K14 and inserted into the HindIII site of pUC18. The resulting plasmid, pIP1798, was used to sequence 1,080 nt of the insert, including the sequences hybridizing with the 147-bp amplicon.

The sequence contains a 642-bp gene including an ATG start codon preceded, 6 nt upstream, by a putative ribosome-binding site. The calculated free energy of association of the most stable structure between this site and the 3′ terminus of the 16S rRNA is −61.5 kJ · mol⁻¹. This gene, not distinguishable from the recently described satG (22), is similar to those encoding SgA acetyltransferases, satA, vat, vatB, and vatC (54.3, 58.0, 60.0, and 60.1% similarity, respectively). satG encodes a putative 214-amino-acid protein of 23,775 Da similar to xenobiotic acetyltransferases (17). It is most similar to the SgA acetyltransferases, SatA, Vat, VatB, and VatC (48.5, 50.0, 59.9, and 50.9% identical amino acids, respectively).

Most vat-related genes in staphylococcal plasmids are contiguous to and downstream from another streptogramin resistance (Sg^r) gene. The pairs of genes are probably cotranscribed (12). However, analysis of the 270- and 170-nt sequences flanking satG did not suggest the presence of any contiguous Sg^r gene.

A DNA fragment of 885 nt containing satG (nt 98 to 982) was amplified from pIP1798 with a primer (nt 957 to 982) containing a single HaeIII site and a primer (nt 98 to 120) whose sequence was modified to create an EcoRI site. This amplicon was cleaved with HaeIII and EcoRI and inserted between the EcoRI and SmaI sites of the shuttle vector, pOX7 (11). The resulting plasmid, pIP1801, introduced by electroporation into the Staphylococcus aureus recipient, RN4220 (15), conferred resistance to pristinamycin IIA [MICs were as follows: 2 mg · liter⁻¹ for RN4220(pOX7) and 8 mg · liter⁻¹ for RN4220(pIP1801)].

The presence of satG in other strains was tested by Southern hybridization experiments using high stringency conditions (2) and a DNA fragment amplified from satG by PCR with the following pair of primers: satG-F (nt positions 354 to 378 in satG) and satG-R (nt positions 878 to 899 in satG) (accession no. AF153312). DNA hybridizing with satG probe was detected in the 32 strains which did not carry satA, including the strain containing vat-vgb (Table 1). Total cellular DNA of strain KH6 was subjected to agarose gel electrophoresis. The satG and vat-vgb sequences migrated to different positions, suggesting that they are not carried by the same plasmid. None of the 53 different S. aureus strains resistant to streptogramin A and described previously (2) contained DNA hybridizing with satG.

The distribution of the streptogramin resistance genes in the collection of E. faecium studied was clearly different from that found in staphylococci (2). None of the satA or satG genes found to be prevalent among E. faecium strains was found in staphylococci.

Nucleotide sequence accession number.

Sequence data from this study has been registered in the GenBank EMBL Data Library under accession no. AF153312.