New Combinations of Mutations in VanD-Type Vancomycin-Resistant Enterococcus faecium , Enterococcus faecalis , and Enterococcus avium Strains

Abstract

ABSTRACT We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 μg/ml) and teicoplanin (MIC, 64 μg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 μg/ml) and to low levels of teicoplanin (MIC, 4 μg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in d -alanyl- d -lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS D : at T 170 I near the phosphorylation site in NEF1, at V 67 A at the membrane surface in BM4653, at G 340 S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl d -Ala: d -Ala ligase had an IS 19 copy in NEF1, a mutation in the serine (S 185 F) or near the arginine (T 289 P) involved in d -Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P 180 S) involved in d -Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY D , a new insertion sequence, IS Efa9 , belonging to the IS 3 family, resulted in the absence of d , d -carboxypeptidase activity. Strain BM4656 had a functional d -Ala: d -Ala ligase, associated with high levels of both VanX D and VanY D activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS D or vanR D gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4 .