Sir,
Treatment options for vancomycin-resistant Enterococcus (VRE) infections are limited. Daptomycin, a lipopeptide with in vitro bactericidal activity against VRE,1 is commonly used to treat such infections. Over the past 5 years an increasing number of reports have documented the emergence of daptomycin-non-susceptible Enterococcus in patients following treatment with daptomycin,1–3 but little is known about how quickly daptomycin non-susceptibility may emerge in vivo. We describe a case of high-level daptomycin resistance (MIC = 256 mg/L) in Enterococcus faecium following only 9 days of exposure to daptomycin and associated with limited mutations in the bacterial genome. All protocols were approved by the UCLA Institutional Review Board.
A woman with a history of thyroid carcinoma, status post-thyroidectomy, morbid obesity and hypertension was diagnosed with a pancreatic tumour and underwent an elective Whipple resection at our medical centre. Her postoperative course was complicated by persistent fevers, worsening leucocytosis and renal failure, for which she underwent haemodialysis. She was started on piperacillin/tazobactam and initial blood, sputum and urine cultures were negative for bacterial growth. She was also found to have persistent hypocalcaemia secondary to hypoparathyroidism (Figure S1, available as Supplementary data at JAC Online). A CT scan of the abdomen and pelvis on postoperative day 10 showed a new 1.8 cm fluid collection that was concerning for abscess formation. After a 14 day course of intravenous piperacillin/tazobactam she was started on intravenous vancomycin, ciprofloxacin and metronidazole. Repeat CT of the abdomen and pelvis with intravenous contrast on postoperative day 17 revealed multiple intra-abdominal fluid collections. Intra-abdominal drains were placed on postoperative day 19 and vancomycin-resistant E. faecium susceptible to daptomycin (MIC = 2 mg/L, isolate 11-9-40) and Candida albicans were isolated by the laboratory from peripancreatic fluid submitted for bacterial culture. She was started on intravenous 6 mg/kg of daptomycin every 48 h, cefepime and metronidazole. She was placed on total parenteral nutrition (TPN) as a nutritional supplement to her oral intake on postoperative day 21 and a high-calcium bath dialysis regimen. As a result, she developed hypercalcaemia during the next 4 days (Figure S1, available as Supplementary data at JAC Online) that resolved after adjustment of the calcium content in the TPN fluid. Urine collected on postoperative day 29 revealed 50 white blood cells/μL, 45 squamous epithelial cells/μL and grew 100 000 cfu/mL daptomycin-non-susceptible E. faecium (daptomycin MIC >256 mg/L, isolate 11-9-43). On postoperative day 35, a daptomycin-non-susceptible E. faecium (daptomycin MIC = 32 mg/L, isolate 11-9-38) was again isolated from intra-abdominal fluid submitted for bacterial culture. The patient was switched to intravenous tigecycline and caspofungin, her fever improved and she remained afebrile until discharge. A repeat CT scan prior to discharge demonstrated a stable appearance of intra-abdominal fluid collections.
Susceptibility testing was performed on all three isolates using the CLSI reference standard broth microdilution (BMD) method4 in cation-adjusted Mueller–Hinton broth, supplemented with 50 mg/L of calcium for daptomycin testing, at the time of bacterial isolation. MICs were confirmed by BMD and Etest (bioMérieux, Durham, NC, USA) on Mueller–Hinton agar (BBL, Lenexa, KS, USA) directly prior to whole-genome sequencing (WGS). A daptomycin MIC >4 mg/L was considered to indicate non-susceptibility.5 WGS of the three isolates was performed using an Illumina HiSeq 2000 sequencer, as described previously.6
Surprisingly few nucleotide polymorphisms were identified between the sequenced genomes of the three isolates (Table 1). Only four single-nucleotide variants (SNVs) were identified between the daptomycin-susceptible (11-9-40) and daptomycin-non-susceptible (11-9-43 and 11-9-38) isolates. Both daptomycin-non-susceptible isolates harboured an SNV in the cardiolipin gene, although at discrete loci (Table 1). No consistent mutation in cls has been associated with daptomycin non-susceptibility in Enterococcus, but cls polymorphism has been described in all daptomycin-non-susceptible E. faecium6 and Enterococcus faecalis7,8 to date, confirming the role of this phospholipase in daptomycin resistance. We have now noted the same SNV, A38C, in two additional E. faecium with high-level daptomycin MICs (e.g. 192 and >256 mg/L, data not shown). Retrospective analysis of these isolates determined they were not clonally related, as described previously.2
Table 1.
Genetic characteristics of daptomycin-non-susceptible E. faecium as compared with parent daptomycin-susceptible strain 11-9-40
| Isolate |
||||||
|---|---|---|---|---|---|---|
| 11-9-40 | 11-9-43 | 11-9-38 | ||||
| Day isolated | 1 | 9 | 14 | |||
| Source | peripancreatic fluid | urine | peripancreatic fluid | |||
| Daptomycin MIC (mg/L) | 2 | >256 | 32 | |||
| Mutations identified by WGS, as compared with 11-9-40 |
||||||
| Gene | nucleotide | predicted amino acid | nucleotide | predicted amino acid | nucleotide | predicted amino acid |
| Cardiolipin synthetase | WT | — | A38C | N12T | G177T | K59T |
| Hypothetical protein | WT | — | C109T | nonsense mutation | C109T | nonsense mutation |
| Dihydrodipicolinate reductase | WT | — | WT | — | deletion of A356 | frameshift |
| Membrane protein | WT | — | 339 bp deletion | — | 339 bp deletion | |
| Aspartate semi-aldehyde dehydrogenase | WT | — | G416T | A139D | WT | — |
WT, wild-type.
Both daptomycin-non-susceptible Enterococcus harboured a nonsense mutation in a hypothetical protein and a 339 bp deletion in a predicted membrane protein of unknown function. Isolate 11-9-43 harboured an SNV in a gene predicted to encode an aspartate semi-aldehyde dehydrogenase not found in 11-9-38, whereas 11-9-38 harboured a frameshift mutation in a dihydrodipicolinate reductase gene not present in 11-9-43. It is unclear if these mutations contributed to the daptomycin-non-susceptible phenotype of these two isolates, but these genes have not been found by other studies and may relate to plasticity in the enterococcal genome. The WGS data also do not provide insight into differences in gene expression levels between the isolates, which may also contribute to differences in daptomycin MIC.
Similar to our previous findings regarding risk factors associated with the development of daptomycin-non-susceptible Enterococcus, this patient was immunocompromised, had recent surgery, an intra-abdominal pathological process, presence of a nidus of daptomycin-non-susceptible Enterococcus infection and exposure to antibiotics associated with the development of VRE, including third-generation cephalosporins and anti-anaerobic agents.1–3 Several factors may have contributed to the development of daptomycin-non-susceptible Enterococcus in this patient. Three clinical events occurred during the time period between isolation of an E. faecium with a daptomycin MIC of 2 mg/L and one with an MIC of 256 mg/L: the patient was exposed to very high concentrations of cefepime, had significant fluctuations in calcium levels and experienced renal failure. Cephalosporins have been known to be associated with the development of VRE, but further case–control studies need to establish whether they can also be associated with the development of daptomycin-non-susceptible Enterococcus. Similarly, although daptomycin has a calcium-dependent mechanism of action and the in vitro calcium concentration of the test medium significantly impacts the daptomycin MIC, the impact of systemic calcium levels on the development of daptomycin-non-susceptible Enterococcus in vivo remains unknown. A 2-fold reduction in calcium concentration (e.g. 25 mg/L versus 50 mg/L) results in a 2-fold increase in the Enterococcus daptomycin MIC.9 The patient's corrected serum calcium ranged from 65.2 to 116.4 mg/L in the month before isolation of daptomycin-non-susceptible Enterococcus, both significantly higher than those used for in vitro testing. This chronic severe hypocalcaemia may have led to an even lower calcium concentration at the infectious focus (abscesses), which caused a loss of daptomycin activity.10 Patients with end-stage renal disease like our patient have a larger volume of distribution for daptomycin and lower Cmax compared with those achieved in healthy volunteers.11 Thus, the concentrations of daptomycin used in our patient (6 mg/kg every 48 h) may have been relatively low and may induced rapid development of daptomycin non-susceptibility.
The acquisition and emergence of daptomycin resistance among enterococci poses both treatment and infection control challenges. Clinicians should be vigilant that rapid emergence of daptomycin-non-susceptible Enterococcus may occur in the setting of disorders of calcium homeostasis, relatively low doses of daptomycin and end-stage renal disease, which may be associated with limited mutations in the bacterial genome.
Funding
This study was supported by internal funding.
Transparency declarations
T. K. and R. M. H. have received research funding from Cubist Pharmaceuticals. R. T.: none to declare.
Supplementary Material
References
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