Letters in Applied Microbiology ISSN 0266-8254
Postantibiotic effect and postantibiotic sub-MIC effect of LTX-109 and mupirocin on Staphylococcus aureus blood isolates L.D. Saravolatz1, J. Pawlak1, H. Martin1, S. Saravolatz1, L. Johnson1, H. Wold2, M. Husbyn2 and W.M. Olsen2 1 St John Hospital and Medical Center, Grosse Pointe Woods, MI, USA 2 Lytix Biopharma AS, Oslo, Norway
Significance and Impact of the Study: Resistant bacterial infections continue to be a challenge for clinicians. Identification of antibiotics with pharmacodynamic advantages may be beneficial in the treatment of these infections. An antibiotic with a longer postantibiotic effect may be able to be administered less frequently resulting in improved adherence. In this study, a new synthetic antimicrobial peptide, LTX-109, demonstrated a more prolonged time for LTX-109 than mupirocin against methicillin-resistant Staphylococcus aureus.
Keywords antimicrobial peptide, LTX-109, MRSA, Mupirocin, Postantibiotic effect, Staphylococcus aureus. Correspondence Louis D. Saravolatz, 19251 Mack Ave., Suite 335, Grosse Pointe Woods, MI 48236, USA. E-mail: [email protected]
2017/0455: received 8 March 2017, revised 25 July 2017 and accepted 3 August 2017 doi:10.1111/lam.12792
Abstract The development of new synthetic antimicrobial peptides like LTX-109 provides a new class of drugs for the treatment of Staphylococcus aureus infections. We evaluated LTX-109 and mupirocin for pharmacodynamic parameters against 10 methicillin-resistant S. aureus isolates. The postantibiotic effect (PAE) is defined as the length of time that bacterial growth is suppressed following a brief exposure to an antibiotic. We also determined the sub-MIC effect (SME) which measures the direct effect of subinhibitory levels on strains that have not previously been exposed to antibiotics. The postantibiotic sub-MIC effect (PASME) is a combination of the PAE and SME. LTX-109 had an average PAE of 551 h vs 104 h for mupirocin. The PA-SME of LTX-109 ranged from 251 to 933 h as the concentration increased from 02 to 04 times the minimal inhibitory concentration (MIC). The PA-SME range for mupirocin was 093– 258 h. LTX-109, as compared to mupirocin, demonstrated prolonged time of effect for these pharmacodynamic parameters, which supports persistent activity for several hours after the drug is no longer present or is below the MIC. The pharmacodynamic parameters studied here suggest that LTX–109 is less likely than mupirocin to generate resistance to S. aureus.
Introduction LTX-109 is a broad-spectrum, fast-acting bactericidal antimicrobial agent that binds to negatively charged membrane components on the bacterial cell wall, which leads to membrane disruption and cell lysis (Nilsson et al. 2015). LTX-109 is a first-in-class chemically synthesized, small peptide drug that is stable against protease degradation. Nonclinical and clinical studies have shown that topical application of LTX-109 has a good safety profile and a low bioavailability (Nilsson et al. 2015). LTX-109 Letters in Applied Microbiology © 2017 The Society for Applied Microbiology
has demonstrated good activity against Staphylococcus aureus strains that are susceptible and resistant to mupirocin (Saravolatz et al. 2012; Nilsson et al. 2015). The pharmacodynamic variables for LTX-109 have not been defined. These include the PAE, SME, and the PASME which are considered important in defining optimal dosage regimens for antimicrobial agents. The PAE is defined as the period of time before the target organism resumes a normal growth rate after the complete removal of the antibiotic (Craig and Gudmundsson 1996). The SME measures the direct effect of subinhibitory levels of 1
L.D. Saravolatz et al.
LTX-109 PAE and PA-SME
the antibiotic on strains that have not been previously exposed to the antibiotic (Odenholt-Tornqvist 1993). The PA-SME represents the time interval that includes the PAE plus the additional time during which growth is suppressed by sub-MIC concentrations (Odenholt-Tornqvist 1993). Previous in vitro studies of LTX-109 demonstrate excellent activity against S. aureus strains resistant to several classes of drugs (Saravolatz et al. 2012; Nilsson et al. 2015). In this study, we determined the PAE, SME and PA-SME of LTX-109 and mupirocin against 10 bloodstream strains of methicillin-resistant S. aureus. Results and discussion A summary of the results for in vitro experiments are provided in Tables 1 and 2. The MIC and minimal bactericidal concentrations (MBC) for LTX-109 were consistently between 2 and 4 lg ml 1 for all 10 isolates. Mupirocin demonstrated better in vitro activity with MICs of 006–025 lg ml 1 with the exception of isolate D11 which had a mupirocin MIC of more than 512 lg ml 1 which is consistent with high-level mupirocin resistance. Notably, the mean PAE for LTX-109 was 551 h which was more than five times longer than mupirocin’s mean PAE of 104 h. The PA-SME demonstrated an increase as the concentration of antibiotic was increased. The PA-SME of LTX-109 was 251 h when the concentration of LTX-109 was 029 MIC and increased to 933 h when the concentration was 049 MIC. Mupirocin had a PA-SME of 093 h at 029 MIC, which increased to 258 h at 049 MIC. The SME of LTX-109 was 065 h, 139 h, and 417 h at 029, 039, and 049 the MIC, respectively, while the SME of mupirocin was 048 h, 102 h and 180 h at the same concentrations. We were unable to identify a relationship between the pharmacodynamic parameters reported in this study and previously identified resistance to antimicrobial agents among the selected strains (Saravolatz et al. 2012). Our study was limited to only one isolate which was resistant to mupirocin, because the high resistance level would not allow us to perform comparative mupirocin testing. Even though the MICs of mupirocin were lower than the MICs for LTX-109, time for the regrowth of bacteria after being exposed to LTX-109 as calculated by the PAE, SME and PA-SME was much longer as compared with mupirocin. This demonstrated that LTX-109 was more efficacious and hence more favourable than mupirocin. The PA-SME was even more impressive considering this parameter may simulate in vivo drug exposure more accurately than the PAE. Since subinhibitory concentrations of a drug frequently persist between antibiotic dosing, the present data suggest that LTX-109 may be administered less frequently than mupirocin. As the problem with 2
mupirocin resistance grows in the United States, alternative agents need to be considered. It has been recently shown that subinhibitory concentrations of antimicrobial agents such as gentamicin, ciprofloxacin and cefotaxime have the ability to induce multidrug resistance to S. aureus by generation of reactive oxygen species (Bhattacharya et al. 2017). It has also been shown that subinhibitory MICs of antimicrobial agents may induce biofilm formation for certain bacterial species (Aka and Haji 2015). Patients may be exposed to subinhibitory concentrations of antibiotics in a variety of settings. We know that antimicrobial agents in subinhibitory MICs can be found in animal livestock as well as the food industry, thus exposing humans to low concentrations of these agents (Nikolic et al. 2011). Furthermore, clinicians have long recognized that patients may take their prescriptions at intervals less frequently than prescribed or discontinue them prior to completion of the full course. This lack of compliance for prescription of antimicrobial agents also provides an opportunity for human exposure to subinhibitory concentrations. In view of the rapid bactericidal activity of LTX-109, its activity against mupirocin-resistant strains and the longer PAE, SME and PA-SME as compared with mupirocin, LTX-109 demonstrates an in vitro and pharmacodynamic advantage over mupirocin. We acknowledge based on the range of results performed in triplicate that there is variability, but these are consistent with the findings of other laboratories determining these pharmacodynamic parameters (Odenholt et al. 2003). Finally, the selection of antimicrobial agents and their dosing regimens needs to consider information about pharmacodynamic properties such as prolonged PASME that prevents the emergence of resistance to serious pathogens such as S. aureus (Olofsson and Cars 2007). The favourable pharmacodynamic profile of LTX-109 with a prolonged PA-SME supports considering LTX-109 in the armamentarium among topical antistaphylococcal agents. Materials and methods The isolates chosen had previously determined MICs that ranged from 2 to 4 lg ml 1 for LTX-109 and 006 lg ml 1 to more than 512 lg ml 1 for mupirocin (Saravolatz et al. 2012, 2013). The staphylococcal cassette chromosome mec typing of this group included six type II, three type IVa and one type IV. The group contained two USA-600 isolates, four USA-100 isolates and three USA-300 isolates. Our group included one vancomycin intermediate S. aureus (VISA), two daptomycin nonsusceptible S. aureus (DNSSA) and one linezolid-resistant S. aureus (LRSA) isolate. These isolates were selected as common strains causing both community- and hospital-associated infections. The PAE was determined by previously described methods (Craig and Gudmundsson 1996). For the PAE Letters in Applied Microbiology © 2017 The Society for Applied Microbiology
2 2 2 4 2 4 4 4 4 4 4 4 4 4 4 4 4 4 2 2 all results (range)
MBC lg ml 1 LTX 109 63 39 46 853 33 51 66 81 54 33 551
(59–675) (34–46) (41–49) (72–101) (30–39) (49–525) (58–75) (78–84) (47–61) (30–39) (33–853)
PAE LTX-109 (h) Results 032 038 055 045 042 06 093 132 077 08 065
(02–045) (01–055) (05–06) (05–05) (025–06) (055–065) (075–105) (125–14) (05–09) (05–11) (032–132)
038 065 065 288 178 105 118 187 247 103 139
(02–065) (06–07) (05–075) (245–33) (17–195) (105) (11–125) (16–20) (175–29) (08–12) (038–288)
087 083 067 1353 473 293 167 33 118 142 417
(075–1) (075–1) (05–08) (1075–1555) (45–52) (275–33) (135–205) (29–375) (98–1405) (12–16) (067–1353)
195 113 065 17 36 187 55 355 31 202 251
(17–21) (10–13) (03–09) (145–204) (285–4) (16–205) (52–575) (31–395) (30–315) (175–25) (065–55)
PA-SME LTX-109 (h)
SME LTX-109 (h)
305 12 173 1683 745 518 938 593 796 303 617
(285–32) (11–14) (14–215) (1405–1855) (605–82) (46–555) (765–114) (445–685) (655–975) (25–37) (12–1683)
Letters in Applied Microbiology © 2017 The Society for Applied Microbiology
MBC lg ml MUP
013 098 UATD 118 093 098 133 14 093 18 104 (11–125) (08–12) (08–11) (12–15) (12–16) (09–10) (15–19) (–013 to 18)
( 035 to 02) (09–11)
PAE Mupirocin (h) Results 012 (005–025) 063 (006–065) UATD 005 (001–01) 023 (01–05) 153 (135–18) 065 (045–075) 052 (03–075) 012 (005–025) 047 (035–07) 048 (005–153)
SME mupirocin (h)
05 (02–07) 138 (09–165) UATD 062 (025–10) 027 (01–05) 395 (355–425) 112 (10–125) 058 (04–075) 032 (02–04) 048 (03–07) 102 (027–395)
097 (07–12) 278 (21–325) UATD 11 (07–15) 035 (01–06) 672 (565–825) 205 (17–25) 077 (06–09) 075 (06–09) 07 (05–095) 18 (035–672)
058 (035–075) 107 (055–155) UATD 053 (025–075) 022 (005–055) 347 (26–455) 115 (085–165) 013 (005–025) 045 (035–055) 075 (025–11) 093 (013–347)
PA-SME mupirocin (h)
083 (08–085) 223 (13–35) UATD 098 (06–12) 03 (015–045) 588 (44–755) 227 (18–29) 05 (05) 067 (06–075) 088 (065–125) 162 (03–588)
(795–98) (67–72) (1285–1485) (115–135) (1205–1775) (40–61) (17–>24)
(37–42) (16–18) (22–27)
13 (09–15) 408 (305–55) UATD 148 (105–17) 043 (035–05) 773 (65–93) 42 (31–565) 108 (10–125) 125 (115–135) 163 (13–20) 258 (043–773)
402 17 242 > 24 888 697 138 123 1423 498 933
UATD, unable to determine; MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentrations; PAE, postantibiotic effect; SME, sub-MIC effect; PA-SME, postantibiotic sub-MIC effect.
D2 012 8 D9 025 16 D11 >512 >512 D19 012 8 D21 006 8 D25 025 16 DNS6 025 32 DNS7 012 16 LNS10 012 16 NRS17 012 16 Means of all results (range)
MIC lg ml MUP
Table 2 Results of mupirocin in vitro experiments
MIC, minimal inhibitory concentration; MBC, minimal bactericidal concentrations; PAE, postantibiotic effect; SME, sub-MIC effect; PA-SME, postantibiotic sub-MIC effect.
D2 D9 D11 D19 D21 D25 DNS6 DNS7 LNS10 NRS17 Means of
MIC lg ml 1 LTX 109
Table 1 Results of LTX-109 in vitro experiments
L.D. Saravolatz et al. LTX-109 PAE and PA-SME
L.D. Saravolatz et al.
LTX-109 PAE and PA-SME
testing, LTX-109 or mupirocin at two times the MIC was added to tubes containing 20-ml Mueller-Hinton (MH) broth. The tubes were inoculated with the organism in the logarithmic phase of growth to obtain a final concentration of 5 9 106 CFU per ml. Each experiment included two control tubes, one with antibiotic free MH broth and a second tube to insure that the antibiotic removal procedure was effective, this tube contained the antibiotic at a 1 : 1000 dilution. The tubes were incubated at 37°C for 20 min. After the initial incubation, the antibiotic was removed by centrifuging the tubes three times at 3000 g for 10 min, decanting and suspending in warm MH broth. Colony counts were taken before exposure to the antibiotics, immediately after washing (time zero) and every hour until the tubes were visibly turbid. Aliquots were diluted in saline and spiral plated onto blood agar plates. The colonies were counted after 24 h. The results were extrapolated from graphs of viability counts (log10 CFU per ml) vs time. The PAE was defined as PAE = T C where T is the time required for the count of CFU in the test culture to increase by 1 log10 above the count observed at time zero, and C represents the corresponding time for the antibiotic free control. Since previous time kill studies showed that LTX-109 is a rapidly bactericidal agent, we performed the PAE using two times the MIC after a 20-min incubation (Saravolatz et al. 2012). For the SME testing, LTX-109 or mupirocin was added to tubes containing MH broth at concentrations of 029, 039 and 049 the MIC. These three tubes and an antibiotic-free control tube were inoculated with organism and incubated at 37°C. Colony counts were performed at time zero and every hour until turbidity C was reached. The SME was defined as SME = Ts where Ts is the time required for the culture exposed to sub-MIC concentrations to increase 1 log10 above the count observed immediately after dilution, and C is the corresponding time for the unexposed control. To determine the PA-SME, the PAE was performed as described above with 29 MIC of LTX-109 or mupirocin. After the 20-min incubation, the antibiotic was removed and the sample was divided into four tubes, three tubes contained the antibiotic at 029, 039 and 049 the MIC. The fourth tube was the control tube containing only MH broth and no antibiotic. Colony counts were taken every 2–3 h until turbidity was seen. The PA-SME was defined as PAC, where Tpa is the time required for the SME = Tpa cultures previously exposed to antibiotic and then reexposed to different sub-MIC concentrations, to increase by 1 log10 above the count obtained immediately after antibiotic removal and C is the corresponding time for the unexposed control. The results are the average of three separate experiments. 4
Acknowledgements This work was supported by an institutional grant from the Lytix Biopharma AS, Oslo, Norway. Conflict of Interest W.M.O. is a stock holder in Lytix Biopharma AS. All others have no conflict of interest to declare. References Aka, S.T. and Haji, S.H. (2015) Sub-MIC of antibiotics induced biofilm formation of Pseudomonas aeruginosa in the presence of chlorhexidine. Braz J Microbiol 46, 149–154. Bhattacharya, G., Diganta, D., Satadal, D. and Banerjee, A. (2017) Exposure to sub-inhibitory concentrations of gentamicin, ciprofloxacin and cefotaxime induces multidrug resistance and reactive oxygen species generation in methicillin–sensitive Staphylococcus aureus. J Med Micro 66, 762–769. Craig, W.A. and Gudmundsson, S. (1996) Postantibiotic effect. In Antibiotics in Laboratory Medicine, ed. Lorian, V. pp. 296–329. Baltimore, MD: Williams and Wilkins Co. Nikolic, N., Mirecki, S. and Blagojevic, M. (2011) Presence of inhibitory substances in raw milk in the area of Montenegro. Mijekarstvo 61, 182–187. Nilsson, A.C., Janson, H., Wold, H., Fugelli, A., Andersson, K., Hakangard, C., Olsson, P. and Olsen, W.M. (2015) LTX109 is a novel agent for nasal decolonization of methicillin-resistant and sensitive Staphylococcus aureus. Antimicrob Agents Chemother 59, 145–151. Odenholt, I., Lowdin, E. and Cars, O. (2003) Postantibiotic, postantibiotic sub-MIC, and subinhibitory effects of PGE9509924, ciprofloxacin, and levofloxacin. Antimicrob Agents Chemother 47, 3352–3356. Odenholt-Tornqvist, I. (1993) Studies on the postantibiotic effect and the postantibiotic sub-MIC effect of meropenem. J Antimicrob Chemother 31, 881–892. Olofsson, S. and Cars, O. (2007) Optimizing drug exposure to minimize selection of antibiotic resistance. Clin Infect Dis 45, S129–S135. Saravolatz, L.D., Pawlak, J., Johnson, L., Bonilla, H., Saravolatz, L.D.I.I., Fakih, M.G., Fugelli, A. and Olsen, W.M. (2012) In vitro activities of LTX-109, a synthetic antimicrobial peptide, against methicillin-resistant, vancomycin-intermediate, vancomycin-resistant, daptomycin-nonsusceptible, and linezolid-nonsusceptible Staphylococcus aureus. Antimicrob Agents Chemother 56, 4478–4482. Saravolatz, L.D., Pawlak, J., Saravolatz, S. and Johnson, L.B. (2013) In vitro activity of retapumulin against Staphylococcus aureus resistant to various antimicrobial agents. Antimicrob Agents Chemother 57, 4547–4550.
Letters in Applied Microbiology © 2017 The Society for Applied Microbiology