Bad bugs, no drugs and no ESKAPE RESULTS: In vitro experimental data Case reports of prosthetic joint infection caused by VRE with reduced susceptibility to daptomycin Vasant JA, Beckley GM and Albur M University Hospitals Bristol NHS Foundation Trust; North Bristol NHS Trust INTRODUCTION The “ESKAPE” pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumanii, P. aeruginosa and Enterobacter species) are common causes of nosocomial infections, “escaping” the effects of antibacterial drugs. Vancomycin resistant Enterococci (VRE) are of particular concern due to a very limited range of active antimicrobials. Daptomycin is the only available cidal drug for such isolates however daptomycin non-susceptibility is now emerging and in lieu of the availability of any novel agents combination therapy is an attractive option. Recent in vitro work has suggested potential synergy for combinations of daptomycin with aminoglycosides1 and beta-lactams2, however experience of daptomycin in combination with the glycycline agent tigecycline is lacking. CASE 1 A 76 year-old male presented post primary total-knee replacement with a discharging wound. Initial tissues grew methicillin-susceptible Staphylococcus aureus, however tissues from further debridement grew a mixture of vancomycin-susceptible and vancomycin resistant Enterococcus faecium with daptomycin MICs of 4mg/L and 6mg/L respectively and a tigecycline MIC 0.19mg/L. Combination treatment with high-dose daptomycin and tigecycline was commenced for a period of 20 days after metalwork removal at first-stage. The CRP fluctuated between 42 and 96 and subsequent tissues from further surgery (spacer exchange) persistently grew the vancomycin-susceptible Enterococcus faecium strain signifying microbiological failure of cure. (daptomycin MIC rising to 16mg/L), and also grew a Proteus mirabilis. Treatment was changed to linezolid and piperacillin-tazobactam. After a further 4 weeks therapy was changed to oral minocycline and ciprofloxacin and the patient is continuing on a prolonged oral course pending second-stage procedure. 1 1 METHODS Clinical data: We present the clinical and microbiological outcomes of two patients with prosthetic joint infection caused by VRE with reduced susceptibility to daptomycin, both treated with combination high dose daptomycin (10mg/kg) and tigecyline therapy. In vitro data: Bactericidal activity of daptomycin (D) and tigecycline (TG) alone and in combination was assessed by time-kill methodology for the VRE isolates from our two patients and a Enterococcus faecalis control strain (ATCC 29212). The susceptibilities of all three isolates are shown in Table 1. Concentrations used for daptomycin were peak (Cmax) 9.3mg/L, steady-state (Css) 4.0mg/L and trough (Cmin) 2.2mg/L reflecting free drug serum concentrations based on actual patient data from therapeutic drug monitoring. We did not measure tigecycline levels in our patients and hence used a fixed concentration of 0.3mg/L representing a phamacokinetically achievable average free drug concentration. A 44 drug exposure matrix of daptomycin with tigecycline was used along with a growth control (GC). Bactericidality was measured by sampling at the following time points: 0, 2, 4, 6, 12, 24, 48 and 72 hours. Emergence of secondary daptomycin resistance was determined by subculturing onto plates containing daptomycin both 2 and 4 times above the baseline MIC. CASE 2 A 78 year old patient underwent an elective scoliosis repair of L2-L5 with metalwork insertion but had ongoing fevers and a leaking wound post-operatively. MRI spine 3 weeks post surgery showed L3/4 and L4/5 discitis and empirical treatment was commenced with piperacillin-tazobactam escalating to meropenem. The patient returned to theatre twice 4 weeks post original surgery for debridement and wash out. Tissues isolated a vancomycin resistant Enterococcus faecium with a daptomycin of MIC 8mg/L and tigecycline MIC of 0.064mg/L. Therapy was changed to high dose daptomycin and tigecycline for a 23 day period. Despite a decrease in CRP from 246 to 15, repeat debridement 8 weeks post surgery was required clinically. Further tissues did not isolate a VRE however did grow a Pseudomonas aeruginosa and therapy was changed to linezolid and piperacillin-tazobactam. Due to further clinical deterioration the metalwork was removed 18 weeks into admission and tissues unfortunately re-grew a vancomycin resistant Enterococcus faecium. Fig 1a Fig 1b RESULTS: In vitro experimental data Time-kill curves (TKC) of the individual isolates at different drug concentrations are shown in Figures 1a (control), 1b (VRE case 1), and 1c (VRE case 2) with a legend key in Table 2. Mean data of all three isolates is shown in Figure 1d. Daptomycin alone exhibited no bactericidal activity at any concentration. Tigecycline showed modest bactericidality with one log drop at 24 & 48 hours. Based on “classic” interpretive criteria the combination of daptomycin with tigecycline did not produce antibiotic synergy at any combination (Table 3). The area-under-bacterial-kill curve (AUBKC) at 24, 48 and 72 hours showed modest but significantly better antimicrobial activity of tigecycline as compared with that of daptomycin at Ctrough and Css (p<0.05) (Table 3). The AUBKC at 48 and 72 hours showed a modest but significant antimicrobial additive effect for the combination of tigecycline with daptomycin at Cmax (p<0.05) (Table 3).  There was no evidence of antagonism of the daptomycin-tigecycline combination at any concentration based on both “classic” interpretive criteria as well as AUBKC data. There was no evidence for the emergence of secondary daptomycin resistance amongst the three isolates over 72 hours for either monotherapy or combination therapy. Fig 1d Fig 1c Table 1. Antimicrobial susceptibilities of study isolates Table 2. Legend key for TKC graphs DISCUSSION These cases highlight the complexity and challenges of prosthetic joint infection caused by resistant bacteria. Not only are therapeutic options limited, but clinical experience and evidence-base is also lacking. Both our patients were treated with a combination of daptomycin and tigecycline during the course of their illness, with short-term clinical and microbiological response. Our in vitro experimental data supports the likelihood of antimicrobial additivity of the combination therapy with no antagonism or emergence of secondary resistance. Antimicrobial therapy however was modified in both cases for a variety of reasons including secondary infections caused by other pathogens and requirement for an oral switch. The role of surgery and metalwork removal is also key and it therefore very difficult to define the exact end points of clinical or microbiological cure and the role that individual antimicrobials play in these chronic relapsing/recurring infections with variable bacterial flora. Within the published literature there are only a handful of other cases where daptomycin-tigecycline combination therapy has been used for Enterococcal infections reported with varying success rates3,4, and limited other published in vitro data5. More studies in the form of multi-centre prospective controlled studies are urgently needed to address the growing problem of infections caused by multi-drug resistant bacteria including ‘ESKAPE’ group of organisms. Antibiotic ATCC 29212 52277 VRE 1 57412 VRE 2 Ampicillin 1mg/L R Vancomycin 2mg/L >256 mg/L 256 Teicoplanin 0.25mg/L 16mg/L Nitrofurantoin 8mg/L Not done Tigecycline 0.12mg/L 0.19mg/L 0.064mg/L Daptomycin No Data 6mg/L 4 or 6 mg/L Moxifloxacin Zone 32 mm Gentamicin Linezolid S Synercid KEY GC Growth control Dtr Daptomycin trough Dss Daptomycin steady state Dpk Daptomycin peak TG Tigecycline fixed concentration 0.3mg/L DtrTG Daptomycin trough + tigecycline fixed concentration 0.3mg/L DssTG Daptomycin steady state + tigecycline fixed concentration 0.3mg/L DpkTG Daptomycin + tigecycline fixed concentration 0.3mg/L Table3. Mean bactericidality (log kill) and AUBKC of study isolates Abx 4hr log-kill 6-hr log-kill 12hr log-kill 24hr log-kill 48hr log-kill 72hr log-kill AUBKC 24 AUBKC 48 AUBKC 72 GC mean 1.1 1.8 2.3 2.1 189.5 386.0 582.5 Dtr mean 0.6 1.0 183.7 380.2 576.6 Dss mean 0.3 0.7 1.9 2.0 179.8 373.7 568.4 Dpk mean 0.1 0.4 1.7 1.6 173.0 363.4 557.4 TG mean -0.1 -0.4 -1.0 -0.9 142.8 265.5 395.2 DtrTG mean -0.3 -0.5 -1.1 -1.3 -1.2 132.9 248.6 363.6 DssTG mean -1.4 135.0 251.0 368.7 DpkTG mean -2.1 126.9 233.8 340.2 REFERENCES 1) ‘Gentamicin improves the activities of daptomycin and vancomycin against Enterococcus faecalis in vitro and in an experimental foreign-body infection model.’ Furustrand Tafin U et al . Antimicrobial Agents & Chemotherapy. 55(10):4821-7, October 2011. 2) ‘B-lactam combinations with daptomycin provide synergy for against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium’. Smith JR et al. JAC 70(6):1738-43, February 2015 3) ‘Multidrug resistant Enterococcus faecium Endocarditis treated with combination high-dose daptomycin and tigecycline therapy’. Schutt AC and Bohm NM. The annals of pharmacotherapy. 43: 2108-12, December 2009. 4) ‘Linezolid- and vancomycin-resistant Enterococcus faecium endocarditis: successful treatment with tigecycline and daptomycin’. Jenkns I. Journal of Hospital Medicine. 2(5): 343-4. September 2007. 5) ‘In vitro activity of daptomycin in combination with B-lactams, gentamicin, rifampicin and tigecycline’. Hindler JA et al. Antimicrobial Agents & Chemotherapy. 59(7): 4279-88. July 2015.