Mutations in Ribosomal Protein L16 and in 23S rRNA in Enterococcus Strains for Which Evernimicin MICs Differ

Abstract

Mutations in ribosomal protein L16 and in 23S rRNA were investigated in 22 Enterococcus strains of different species and for which the MICs of evernimicin differ (MICs, 0.023 to 16 μg/ml). Amino acid changes (Arg56His, Ile52Thr, or Arg51His) in protein L16 were found in seven strains, and a nucleotide G2535A mutation in 23S rRNA was found in 1 strain among 13 for which the MICs are ≥1 μg/ml.

Evernimicin (SCH-27899) (EVN) is an oligosaccharide antibiotic with excellent activity against a broad range of gram-positive pathogenic bacteria (7, 12). This antibiotic inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit (5, 9). Different mechanisms of EVN resistance have been reported to date: (i) mutations in the rplP gene that encodes the ribosomal protein L16 (detected in Streptococcus pneumoniae [3], Enterococcus faecalis and Enterococcus faecium animal isolates [1], and mutants of Staphylococcus aureus [10]; (ii) mutations in domain V of 23S rRNA gene (detected in S. pneumoniae [2] and Halobacterium halobium isolates [5]); and (iii) synthesis of an rRNA methyltransferase (EmtA) that confers high-level EVN resistance (detected in an E. faecium animal strain [8]). The number of rRNA operons (rrn) in enterococci has been previously estimated between five and six, depending upon the species (6, 11).

The objective of this study was to analyze the sequences of the ribosomal L16 proteins and of the 23S rRNA genes of 19 Enterococcus strains that were chosen from our collection of strains on the basis of their increased EVN MIC (MIC range, 0.75 to 16 μg/ml). Enterococcus strains were recovered from feces of healthy animals (12 E. faecium strains, two E. faecalis strains, four Enterococcus hirae strains, and one Enterococcus durans strain). Three additional strains (one strain each of E. faecium, E. faecalis, and E. hirae) for which the MICs of EVN were in the range 0.023 to 0.19 μg/ml were also included as control susceptible strains. All 22 strains in this study showed an unrelated pulsed-field gel electrophoresis pattern after SmaI digestion (data not shown). MICs of EVN were determined by the E-test method following the manufacturer's guidelines (AB Biodisk, Solna, Sweden).

A fragment of 414 bp of the gene that encodes the ribosomal protein L16 and a 634-bp fragment of the domain V of the 23S rRNA gene that includes the peptidyltransferase region were amplified by PCR in all 22 strains using primers previously described (1, 13). Amplicons were purified and sequenced in both strands using the same primers as those used for PCRs. The 23S rRNA gene was numbered following Escherichia coli position numbering. The nucleotide sequences obtained for both genes were compared with the reference sequences indicated in Table 1.

TABLE 1.

Sequences used as reference for the study of mutations in the rplP gene that encodes the ribosomal protein L16 and in the 23S rRNA gene

Enterococcus species	Reference protein L16 (rplP gene)		Reference 23S rRNA gene
	Species	GenBank accession no.	Species	GenBank accession no.
E. faecium	E. faecium	AF291861	E. faecium	AJ295305
E. faecalis	E. faecalis	EF0213a	E. faecalis	AJ295306
E. hirae	E. faecium	AF291861b	E. hirae	AJ295309
E. durans	E. faecium	AF291861b	E. durans	AJ295304

^aLocus name per The Institute for Genomic Research (www.tigr.org).

^bThe sequence for the rplP gene of E. hirae and E. durans species was not found in the GenBank. For this reason, the sequence reported for E. faecium (AF291861) was used as reference for detection of mutations in both species.

Results of EVN MICs as well as the mutations detected in both rplP and 23S rRNA genes in the 22 strains of this study are shown in Table 2.

TABLE 2.

Mutations in the rplP gene that encodes the ribosomal L16 protein and in the 23S rRNA gene in Enterococcus strains for which the MICs of EVN differ

Species	Strain	MIC of EVN by E-test (μg/ml)	Ribosomal rplP gene		23S rRNA geneb
			Deduced amino acid changes in L16 proteind	Silent mutation(s)
E. faecium	Z-277e	0.023
	Z-247	0.75		A129T, T327G, A339G
	Z-185	0.75
	Z-166	0.75		C324G
	Z-177	0.75
	Z-145	0.75
	Z-181	1
	Z-100	1	Arg 56 (CGT)→His (CAT)
	Z-22	1.5	Arg 56 (CGT)→His (CAT)
	Z-150	1.5	Ile 52 (ATC)→Thr (ACC)
	Z-105	2	Arg 56 (CGT)→His (CAT)
	Z-32	3	Arg 56 (CGT)→His (CAT)
	Z-214	3		C69T, A72T, A129T, A339G	-c
E. faecalis	Z-68e	0.19			G2581A
	Z-123	8	Arg 51 (CGT)→His (CAT)	C111T	G2581A
	Z-37	16			G2581A, G2535A
E. hiraea	Z-87e	0.19	Pro 110 (CCT)→Ser (TCT)	T63C, C69T, A72T, A129T, C318T, T327G, A339G
	Z-246	0.75	Pro 110 (CCT)→Ser (TCT)	T63C, C69T, A72T, A129T, C318T, T327G, A339G
	Z-96	1	Pro 110 (CCT)→Ser (TCT)	T63C, C69T, A72T, A129T, C318T, T327G
	Z-98	2	Pro 110 (CCT)→Ser (TCT)	T63C, C69T, A72T, A129T, C318T, T327G
	Z-89	2	Pro 110 (CCT)→Ser (TCT)	T63C, C69T, A72T, A129T, C318T, T327G, A339G
			Arg 56 (CGT)→His (CAT)
E. duransa	Z-203	2		C69T, A72T, A129T, A339G

^aSequences obtained for the rplP gene that encodes the L16 protein in isolates of this species were compared to the E. faecium sequence (AF291861).

^bThe 23S rRNA gene was numbered according to E. coli position numbering (GenBank accession no. J01695).

^cThis strain showed the mutation A2211G (identical to that in GenBank sequence X79341).

^dAmino acids are identified by number, with the triplet shown parenthetically. parenthetically.

^eControl.

E. faecium.

No amino acid substitutions in ribosomal protein L16 were predicted from the sequences obtained from the six E. faecium strains for which the MIC of EVN was ≤ 0.75 μg/ml. Nevertheless, amino acid changes (Arg56His and Ile52Thr) were predicted in five of seven strains for which the MICs of EVN were in the range 1 to 3 μg/ml. These changes in protein L16 had been previously reported in E. faecium isolates for which the MICs of EVN were ≥3 μg/ml (1). Different silent mutations in the rplP gene were also identified in three of the analyzed strains (Table 2).

Twelve of the 13 strains showed the same wild-type sequence as that previously reported (GenBank accession no. AJ295305) when 23S rRNA amplicons were sequenced and analyzed. One of the strains showed the mutation A2211G (Table 2), but this mutation was included in another previously published sequence (GenBank accession no. X79341). The position 2211 is located in hairpin 79 of domain V of the 23S rRNA gene, and this place is far away of the binding site of EVN to the 50S ribosomal subunit (5).

E. faecalis.

The mutation encoding the Arg51His change was observed in L16 protein of the strain Z-123 (MIC, 8 μg/ml). To our knowledge, the Arg51His substitution has not been described to date in Enterococcus and also has not been reported as a spontaneous change in any microorganism. Position 51 of protein L16 seems to be important for EVN action. The Arg51His or Arg51Cys changes have been previously obtained by chemical mutagenesis in S. aureus, causing a marked increase in the MIC of EVN (30 or 60 times, respectively) with respect to the wild-type strain (10). In addition, the Arg51Cys change has also been reported in S. pneumoniae mutants obtained by site-directed mutagenesis (3). No predicted amino acid changes in protein L16 were identified in strains Z-68 and Z-37 (EVN MICs, 0.19 and 16 μg/ml, respectively) (Table 2).

Clear single-nucleotide peaks were obtained in all chromatograms when the 23S rRNA amplicons were sequenced, and that suggests the presence of the same sequence in all fragments of rrn operons in our strains. A nucleotide mutation G2581A (with respect to the sequence AJ295306) was identified in the 23S rRNA amplicons of our three E. faecalis strains. The nucleotide at position 2581 is far from the EVN binding site in the ribosomal 50S subunit (4, 5), and thus, a mutation in this nucleotide could not be related to the increase in the MIC of EVN. A G2535A transition in the 23S rRNA gene was detected in E. faecalis isolate Z-37 (MIC, 16 μg/ml). This point mutation causes the disappearance of a target for the AvaII enzyme. A wild-type gene should give rise to three fragments (552, 72, and 10 bp) while a G2535A-mutated gene would yield two fragments (624 and 10 bp). The analysis of the restriction fragment length polymorphism pattern of the amplicon after AvaII digestion suggests that all copies of the 23S rRNA gene are mutated in the Z-37 strain (data not shown). This nucleotide mutation has been associated with an increase in the MIC of EVN for isolates of S. pneumoniae (2) and H. halobium (5), but never before for Enterococcus. Other nucleotide mutations in 23S rRNA gene (at positions 2536, 2469, and 2480) associated with increased MICs or EVN for S. pneumoniae isolates have also been reported (2).

E. hirae.

Five E. hirae strains were analyzed in this study; for four of these the MICs of EVN were in the range 0.75 to 2 μg/ml, and there was one susceptible control strain for which the MIC was 0.19 μg/ml. The sequences of the rplP gene have been compared to that from E. faecium (Table 1). All five strains studied, including the very susceptible one, showed a predicted Pro110Ser change in the protein L16. Thus, this change does not seem to be related to an increase in the MIC of EVN. A second amino acid change (Arg56His) was predicted in E. hirae Z-89 strain (MIC, 2 μg/ml). Different silent mutations were detected in the rplP gene that encodes protein L16 in all E. hirae isolates. No nucleotide mutations were identified in the partial 23S rRNA gene sequenced of the 5 E. hirae strains studied.

E. durans.

No amino acid changes in L16 ribosomal protein (compared to wild-type L16 protein of E. faecium AF291861) and no nucleotide mutations in the 23S rRNA gene were identified in the single E. durans isolate analyzed in this study (MIC of EVN, 2 μg/ml).

As a conclusion, an Arg51His change in the L16 protein and a G2535A mutation in the 23S rRNA gene have been detected in two E. faecalis strains, and these mutations could be associated with the high MICs of EVN (8 and 16 μg/ml) for these strains. To our knowledge, this is the first time that these mutations have been reported in Enterococcus. Amino acid changes in the L16 protein or nucleotide mutations in the 23S rRNA gene were detected as being a presumable EVN resistance mechanism in 8 strains among 13 for which the MICs were ≥1 μg/ml. Other mechanisms of resistance (such as the EmtA rRNA methyltransferase) should be sought in the remaining 5 strains for which the MICs of EVN are increased.