In Vitro Activity of the Novel Pleuromutilin Lefamulin (BC-3781) and Effect of Efflux Pump Inactivation on Multidrug-Resistant and Extensively Drug-Resistant Neisseria gonorrhoeae

ABSTRACT

We evaluated the activity of the novel semisynthetic pleuromutilin lefamulin, inhibiting protein synthesis and growth, and the effect of efflux pump inactivation on clinical gonococcal isolates and reference strains (n = 251), including numerous multidrug-resistant and extensively drug-resistant isolates. Lefamulin showed potent activity against all gonococcal isolates, and no significant cross-resistance to other antimicrobials was identified. Further studies of lefamulin are warranted, including in vitro selection and mechanisms of resistance, pharmacokinetics/pharmacodynamics, optimal dosing, and performance in randomized controlled trials.

TEXT

Neisseria gonorrhoeae has developed resistance to all antimicrobials used in monotherapy of gonorrhea (1). Many gonorrhea treatment guidelines currently recommend ceftriaxone 250 to 500 mg × 1 plus azithromycin 1 to 2 g × 1 as empirical first-line treatment (2–6). However, recently the first global treatment failure with recommended dual antimicrobial therapy was verified (7), and new antimicrobials for treatment are crucial (1).

Lefamulin (BC-3781; Nabriva, Vienna, Austria) is a novel semisynthetic pleuromutilin (8, 9; S. Paukner, A. Gruss, T.R. Fritsche, Z. Ivezic-Schoenfeld, and R. N. Jones, poster E-1183, presented at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 2013) which inhibits bacterial protein synthesis and growth by binding to the peptidyltransferase center of the 50S ribosomal subunit (interfering with 23S rRNA) with high affinity and specificity and uses different targets than do other antimicrobial classes (9). Lefamulin has completed a phase II clinical randomized controlled trial (RCT) for acute bacterial skin and skin structure infections and is currently undergoing two phase III RCTs for the treatment of community-acquired bacterial pneumonia (10–13).

We evaluated the in vitro activity of lefamulin against clinical gonococcal isolates and international reference strains (n = 251) and the effect of inactivation of efflux pumps (MtrCDE, MacAB, and NorM). The tested gonococcal isolates were diverse geographically (having essentially global representativeness), temporally (1991 to 2016), and phenotypically/genetically, including 34 international reference strains (all WHO reference strains [14, 15]), 100 consecutive clinical Swedish gonococcal isolates from 2016, and 117 isolates selected for their resistance phenotype. The latter included a high proportion (49%) of azithromycin-resistant isolates (macrolides also target the 50S ribosomal subunit [23S rRNA]), extensively drug-resistant (XDR [16]) isolates (7, 17–19), and numerous isolates with in vitro or clinical resistance to extended-spectrum cephalosporins (ESCs) and other high-level clinical resistance and multidrug resistance (MDR [16]) to other antimicrobials previously used for gonorrhea treatment.

The MICs of lefamulin were determined by the CLSI agar dilution technique, and MICs of ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, ampicillin, and tetracycline were determined by the Etest (AB bioMérieux, Marcy l'Etoile, France). The mtrD, macA, and norM genes, coding for (sub)components of the respective efflux pumps, were inactivated in WHO F, WHO O, WHO P, and WHO X (14), and one clinical strain with high-level azithromycin resistance (HLAziR; azithromycin MIC ≥256 μg/ml), as previously described (20).

Lefamulin showed potent activity against all 251 N. gonorrhoeae isolates (Table 1). Briefly, the modal MIC, MIC₅₀, and MIC₉₀ values and the MIC range of lefamulin were low, i.e., 0.5 μg/ml, 0.25 μg/ml, 1 μg/ml, and 0.004 to 2 μg/ml, respectively (Table 1).

TABLE 1.

Antimicrobial activities of lefamulin against all Neisseria gonorrhoeae isolates and isolate subgroups and susceptibility of all isolates to antimicrobials currently or previously recommended for treatment of gonorrhea

Antimicrobial and isolate group (no.)	MIC (μg/ml)a				Susceptibility (%)b
	Range	50%c	90%d	Modal	S	I	R
Lefamulin
All isolates (251)	0.004–2	0.25	1	0.5	NDe	ND	ND
Contemporary consecutive isolates (100)f	0.004–1	0.25	0.5	0.25	ND	ND	ND
Selected resistant isolates (117)	0.016–2	0.5	1	0.5	ND	ND	ND
Azithromycin-resistant isolates (123)	0.016–2	0.5	1	0.5	ND	ND	ND
Azithromycin-susceptible isolates (128)	0.004–1	0.25	0.5	0.5	ND	ND	ND
Reference strains (34)	0.016–2	0.5	1	0.5	ND	ND	ND
Ceftriaxone (251)	<0.002–4	0.008	0.064	0.004	97.2	ND	2.8
Cefixime (251)	<0.016–8	0.016	0.125	<0.016	91.2	ND	8.8
Azithromycin (251)	0.016–>256	0.5	16	16	40.6	10.4	49.0
Spectinomycin (251)	4–1,024	16	16	16	98.0	ND	2.0
Ciprofloxacin (251)	<0.002–>32	0.25	>32	≥32	48.6	0.0	51.4
Ampicillin (251)	0.016–>256	0.5	8	0.125	37.8	47.4	14.7
Tetracycline (251)	0.125–128	2	16	2	25.9	23.1	51.0

^aMIC was determined using the agar dilution technique for lefamulin and the Etest for the additional antimicrobials. Only whole MIC dilutions are reported.

^bS, susceptible; I, intermediately susceptible; R, resistant. The EUCAST breakpoints (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_7.0_Breakpoint_Tables.pdf) were applied for all antimicrobials.

^cMIC at which 50% of the isolates tested were inhibited.

^dMIC at which 90% of the isolates tested were inhibited.

^eNot determined due to lack of interpretative criteria.

^fConsecutive clinical Swedish gonococcal isolates obtained in 2016.

No significant cross-resistance between lefamulin and the macrolide azithromycin was identified. A total of 123 (49%) azithromycin-resistant isolates, 8 of these with azithromycin MICs of >256 μg/ml, were compared to 128 (51%) azithromycin-sensitive isolates, and the modal MIC, MIC₅₀, MIC₉₀, and MIC range of lefamulin for these groups did not show significant differences (Table 1). The MIC distributions for lefamulin and azithromycin (Fig. 1a) and a comparison of the MIC values also showed a relatively limited correlation (Fig. 1b).

FIG 1.

(a) MIC distribution for the novel pleuromutilin lefamulin and the macrolide azithromycin, including clinical Neisseria gonorrhoeae isolates (n=217) and international reference strains (n=34). (b) Comparison of the MICs of azithromycin and lefamulin for the identical material.

The isolates resistant and susceptible to also the nonmacrolide antimicrobials had similar low lefamulin MICs, and no cross-resistance with lefamulin was identified. For the isolates with ESC resistance and isolates resulting in verified ESC treatment failures (n = 22, 8.8%), including failures caused by “superbugs” such as H041 (18) and F89 (17, 19), the lefamulin MICs were 0.5 μg/ml (n = 16) and 1 μg/ml (n = 6).

Inactivation of the MtrCDE efflux pump in all five strains examined decreased the lefamulin MICs significantly (4- to 6-fold). Inactivation of the MacAB or NorM efflux pump had no significant impact on the lefamulin MICs (Table 2).

TABLE 2.

MIC of lefamulin in Neisseria gonorrhoeae wild-type strain and strains with the MtrCDE, MacAB, or NorM efflux pump inactivated

Strain	MIC (μg/ml) of:
	WTa	MtrCDE inactivated	MacAB inactivated	NorM inactivated
WHO F	0.125	0.008	0.064	0.064
WHO O	0.5	0.016	0.5	0.5
WHO P	2	0.032	2	2
WHO X	0.5	0.016	0.5	0.5
HLAziR	1	0.064	1	1

^aWT, wild type.

The novel pleuromutilin lefamulin had potent in vitro activity against a large and geographically, temporally, and genetically diverse collection of clinical gonococcal isolates and international reference strains, including high-level resistant MDR and XDR isolates. No significant cross-resistance between lefamulin and any other antimicrobials, including the macrolide azithromycin, was identified.

The MtrCDE efflux pump influenced susceptibility to lefamulin, as reported for several antimicrobial classes (20). Further studies on the effect of the MtrCDE efflux pump on lefamulin activity and in vitro studies selecting for resistance to lefamulin, using subinhibitory lefamulin concentrations, detailing the resistance determinants in gonococci and investigating the fitness of selected resistant mutants would be valuable to predict the future emergence and transmission of lefamulin resistance.

Lefamulin has finished a successful phase II RCT as a treatment option for acute bacterial skin and skin structure infections and is undergoing two phase III RCTs for treatment of community-acquired bacterial pneumonia (10–13). It has also been shown in phase I clinical trials including healthy subjects that oral and intravenous lefamulin, at exposures predicted to be therapeutically efficacious, is safe and well tolerated, with no significant age, weight, or gender-related differences (W. T. Prince, W. W. Wicha, D. B. Strickman, V. Moschetti, F. Obermayr, and R. Novak, abstr. P906, presented at the 20th European Congress of Clinical Microbiology and Infectious Diseases, 2010; W. W. Wicha, C. Lell, D. K. Logan, and W. T. Prince, abstr. A1-0192010, presented at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2010). Lefamulin appears to be well tolerated orally and as an injection, with only some local signs and mild, self-limiting symptoms related to the infusion site after the administration of 100 or 150 mg intravenously every 12 h over 5 to 14 days (13). Pharmacokinetic studies have shown rapid penetration of lefamulin into skeletal muscle tissue, subcutaneous adipose tissue, and epithelial lining fluid (21, 22; S. M. Bhavnani, P. G. Ambrose, W. W. Wicha, Z. Ivezic-Schoenfeld, W. T. Prince, C. M. Rubino, abstr. 793, presented at ID Week, 2015). Significant distribution of lefamulin to tissues relevant for sexually transmitted infections (STIs) has also been shown in animals (unpublished data). However, additional pharmacodynamic and pharmacokinetic studies (including evaluation of dosing) and/or modeling, particularly for human urogenital, pharyngeal, and anorectal mucosa, remain imperative to assess lefamulin as a treatment option for gonorrhea. No in vitro antagonism of lefamulin in combination with other examined antimicrobials was observed in a study including selected Gram-positive and Gram-negative bacteria (S. Paukner, A. Stoneburner, Z. Ivezic-Schoenfeld, and C. Pillar, abstr E-1161, presented at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 2013). Although no STI agents were tested, this is promising for potential future dual antimicrobial therapies for gonorrhea and possibly also for Mycoplasma genitalium infections.

In conclusion, lefamulin appears promising for treatment of gonorrhea. However, for lefamulin to become a first-line drug for gonorrhea, lefamulin should have high efficacy (cure >95% of urogenital and extragenital infections), perform well in a combination therapy (to delay resistance development), be widely available and affordable in appropriate quality and dose, have no or minimal drug-drug interactions and low toxicity, be well tolerated, and ideally have activity also against concurrent Chlamydia trachomatis and M. genitalium infections (S. Paukner, A. Gruss, T.R. Fritsche, Z. Ivezic-Schoenfeld, and R. N. Jones, Poster E-1183, presented at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 2013). Lefamulin fulfills many of these criteria, and further studies are warranted.