Dataset Information

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus β-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

ABSTRACT:

Background

The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown.

Methods

We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and β-lactam binding affinity in HOU503 versus R497.

Results

DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased β-lactam binding affinity of PBP5 of HOU503 compared with that of R497.

Conclusions

Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus β-lactam therapy.

SUBMITTER: Kebriaei R

PROVIDER: S-EPMC7529040 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/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 IS19 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, ISEfa9, belonging to the IS3 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.