In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens

ABSTRACT

The pleuromutilin antibiotic lefamulin demonstrated in vitro activity against the most relevant bacterial pathogens causing sexually transmitted infections (STI), including Chlamydia trachomatis (MIC_50/90, 0.02/0.04 mg/liter; n = 15), susceptible and multidrug-resistant Mycoplasma genitalium (MIC range, 0.002 to 0.063 mg/liter; n = 6), and susceptible and resistant Neisseria gonorrhoeae (MIC_50/90, 0.12/0.5 mg/liter; n = 25). The results suggest that lefamulin could be a promising first-line antibiotic for the treatment of STI, particularly in populations with high rates of resistance to standard-of-care antibiotics.

TEXT

Despite the availability of antibiotics, the management of sexually transmitted infections (STI) has become a significant global health challenge, with 131 million new chlamydia and 78 million gonorrhea infections globally (1, 2). Additionally, Mycoplasma genitalium is considered as significant as Chlamydia trachomatis, with a prevalence of 10 to 45% in men with nongonococcal urethritis and 1 to 3.3% in the general population (3). Their control relies on effective antibiotic therapy, but STI pathogens are progressively developing resistance. Neisseria gonorrhoeae organisms resistant to all of the recommended antibiotics have been reported globally, leaving injectable ceftriaxone as the only remaining option for empirical first-line therapy (4–6). Notably, even ceftriaxone resistance has started to emerge, leaving very few treatment options for gonorrhea (7). Similarly, M. genitalium is evolving into an untreatable infection, with a rapidly increasing proportion of multidrug-resistant strains spreading primarily in the Asia-Pacific region (3, 8–11). In patients failing treatment with azithromycin, moxifloxacin, and doxycycline, no other effective antimicrobials are available. Although treatment with pristinamycin has been tried with some success, failure with this treatment is common (12, 13). Until now, resistant C. trachomatis cases are considered to be rare and unconfirmed. The WHO and national authorities have issued action plans to prevent the spread and impact of antimicrobial resistance in STI and recommend dual therapy, but new antibacterial agents are needed (14). Since patients with STI are commonly managed syndromically, therapies should target the relevant etiologies, and antimicrobial agents with both oral and intravenous formulations are preferable (15).

Lefamulin is a new antibiotic belonging to the pleuromutilin class of antibiotics selectively inhibiting bacterial translation by binding to the peptidyl transferase center (PTC) of the bacterial ribosome via four H bonds and other interactions at the A- and P-site, whereby nucleotides in the PTC shift and tighten the binding pocket around lefamulin (16–18). This unique mechanism of action minimizes the potential for cross-resistance with other antibacterial agents currently in use and is assumed to be the reason for the low potential for the development of pleuromutilin resistance (19). However, pleuromutilin resistance, though sporadic, has been shown to be mediated by plasmid-encoded or chromosomal resistance determinants such as cfr or vgaA-vgaB, and caution is indicated to prevent resistance from spreading in the clinical setting. Lefamulin has been studied after intravenous and oral administrations in adult patients with acute bacterial skin and skin structure infections (phase 2) and with community-acquired bacterial pneumonia (phase 3) (20–23). Lefamulin showed good tissue penetration, particularly into the epithelial lining fluid (24–27) and into tissues relevant for STI, including prostate compartments and pelvic tissues (W. W. Wicha, C. Henson, and K. Webbley, unpublished data). In the present study, we evaluated the in vitro activity of lefamulin and comparator antibiotics against the most prevalent bacterial pathogens causing STI, including C. trachomatis, M. genitalium, and gonococci.

Lefamulin (BC-3781), provided by Nabriva Therapeutics (Vienna, Austria), was dissolved in water and further diluted in culture medium. Comparators were obtained from commercial sources: azithromycin (Groton Laboratories, Pfizer Inc., Groton, CT, USA), clarithromycin, erythromycin, and doxycycline (Sigma-Aldrich, Germany, Denmark), moxifloxacin and ciprofloxacin (Bayer, Germany), and cefixime, ceftriaxone, and penicillin (Sandoz, Austria).

C. trachomatis serovars (one strain per serovar, n = 15) L₂/434 (ATCC VR-902B), L₃/404 (ATCC VR-903), A/G-17, B/TW-5, Ba/AP-2 (ATCC VR-347), E/UW-5, G/UW-57 (ATCC VR-878), and SA₂f were cultured in McCoy cells (ATCC CRL-1696), and the serovars C/TW-3 (ATCC VR-578), D/UW-3 (ATCC VR-885), F/MRC 301, H/UW-4/Cx, I/UW-12/Ur (ATCC VR-880), J/UW-36 (ATCC VR-886), and K/UW-31 (ATCC VR-887) were cultured in HeLa 229 cells (ATCC CCL-2.1). Strains were obtained from ATCC (Manassas, VA, USA) or were clinical isolates. MIC determinations were performed on monolayers of the cells infected with C. trachomatis (10² to 10³ inclusion forming units [IFU] per 3 × 10⁵ cells) on glass coverslips at drug concentrations ranging from 2.56 to 0.0003 mg/liter (28). After incubating at 35°C (5% CO₂) for 48 to 72 h, the cells were fixed in methanol, and inclusions were stained with an alcoholic iodine solution. The MIC was defined as the lowest concentration of antibiotic at which no inclusion was observed.

Susceptibility testing of M. genitalium G37^T (ATCC 33530) and the multidrug-resistant M. genitalium isolates (M6489, M6711, M6712, M6714, and M6735) obtained from five patients who failed treatment with azithromycin, moxifloxacin, and doxycycline was performed using the Vero cell culture and a quantitative real-time PCR method (29). Macrolide resistance-mediating mutations were determined by sequencing as previously described (30).

Susceptibility testing of N. gonorrhoeae clinical isolates (n = 25) was performed by the agar dilution technique described by the Clinical and Laboratory Standards Institute (31). N. gonorrhoeae F-18 (ATCC 49226) was included as a reference strain.

Lefamulin and comparators were tested against 15 different serovars of the intracellular pathogen C. trachomatis. These included the nondisseminating serovars D to K that cause oculogenital infection (32), serovars A, B, Ba, and C that cause ocular trachoma (33), and the invasive lymphogranuloma venereum serovars L2, L3, and SA2f associated with systemic spread to the regional lymph nodes. Lefamulin exhibited activity against all of the C. trachomatis strains being inhibited at a lefamulin concentration of ≤0.04 mg/liter (Table 1). The activity of lefamulin (MIC_50/90 of 0.02/0.04 mg/liter) was comparable to those of doxycycline (MIC_50/90 of 0.01/0.02 mg/liter), moxifloxacin (MIC_50/90 of 0.05/0.05 mg/liter), and clarithromycin (MIC_50/90 of 0.005/0.01 mg/liter) and was 5-fold greater than that of azithromycin (MIC_50/90 of 0.1/0.2 mg/liter) or erythromycin (MIC_50/90 of 0.1/0.2 mg/liter). The high activity of lefamulin against this intracellular organism is in line with the high intracellular concentrations observed in vitro (24).

TABLE 1.

MIC_50/90 and range values of lefamulin and comparator antibiotics against C. trachomatis, N. gonorrhoeae, and M. genitalium

Antibiotic	No. of isolates	MIC (mg/liter)
		Range	50%	90%
C. trachomatis, serovars A to Sa
Lefamulin	15	0.01 to 0.04	0.02	0.04
Linezolid	15	1.6 to >12.8	12.8	>12.8
Azithromycin	13	0.025 to 0.2	0.1	0.2
Clarithromycin	13	0.0006 to 0.01	0.005	0.01
Doxycycline	13	≤0.005 to 0.02	0.01	0.02
Erythromycin	13	0.025 to 0.4	0.1	0.2
Moxifloxacin	13	≤0.025 to 0.05	0.05	0.05
M. genitalium, including multidrug-resistant strainsb
Lefamulin	6	0.002 to 0.063	0.063	NAc
Azithromycin	6	0.008 to >16	>16	NA
Erythromycin	6	0.125 to ≥64	≥64	NA
Doxycycline	6	0.5 to 1	1	NA
Moxifloxacin	6	0.125 to >16	8	NA
Ciprofloxacin	6	8 to >16	16	NA
N. gonorrhoeae
Lefamulin	24	0.03 to 1	0.12	0.5
Azithromycin	24	0.03 to 0.5	0.12	0.5
Cefixime	24	≤0.008 to 0.06	0.015	0.03
Ceftriaxone	24	≤0.008 to 0.03	≤0.008	0.03
Ciprofloxacin	24	≤0.008 to >4	0.008	>4
Gentamicin	24	1 to 8	8	8
Penicillin	24	≤0.015–>32	0.25	>32
Tetracycline	24	0.12 to 4	0.5	4

^aC. trachomatis serovars were A, B, Ba, C, D, E, F, G, H, I, J, K, L2, L3, SA2f (LGV lab strain). MIC was tested in HeLa229 and McCoy cells.

^bM. genitalium strains included 5 multidrug-resistant strains and the G37 type strain ATCC 33530.

^cNA, not applicable.

All M. genitalium strains were inhibited at a lefamulin concentration of ≤0.063 mg/liter (Table 1). The M. genitalium strains included multidrug-resistant isolates highly resistant to fluoroquinolones due to mutations in the parC gene (moxifloxacin and ciprofloxacin, MIC₅₀s of 8 and 16 mg/liter, respectively) and to macrolides (erythromycin and azithromycin, MIC₅₀s of ≥64 and >16 mg/liter, respectively) conferred by single point mutations at positions A2058 or A2059 in the 23S rRNA gene (see Table S2 in the supplemental material). The MIC value for doxycycline was 1 mg/liter despite the clinical and microbiological treatment failures in some of the patients from whom the strains were isolated. The reason for the discrepancy between the in vitro susceptibility of these strains to doxycycline and the lack of in vivo treatment efficacy remains unclear. However, because of this low clinical efficacy, doxycycline is not recommended as a first-line treatment against M. genitalium (11). Even for these multidrug-resistant isolates, lefamulin displayed potent activity, with MICs ranging from 0.016 to 0.063 mg/liter, and was consistently the most active compound tested (Table 1). Further investigations against a larger set of M. genitalium strains and other mycoplasma species are warranted.

Lefamulin demonstrated activity against N. gonorrhoeae (MIC_50/90 of 0.12/0.5 mg/liter), equivalent to that observed with azithromycin (MIC_50/90 of 0.12/0.5 mg/liter) and 4-fold greater activity than tetracycline against susceptible strains (MIC₅₀ of 0.5 mg/liter). Lefamulin's in vitro activity was unchanged regardless of the presence or absence of the resistance to the fluoroquinolones, tetracyclines, or penicillins (Table 1). The resistance rates among the tested N. gonorrhoeae strains were 12.5% for ciprofloxacin, 29.2% for tetracycline, and 20.8% for penicillin when breakpoints as published by CLSI were applied (34). All tested N. gonorrhoeae isolates were fully susceptible to ceftriaxone (MIC_50/90 of ≤0.008/0.03 mg/liter) and cefixime (MIC_50/90 of 0.015/0.03 mg/liter) and were susceptible or intermediate susceptible to azithromycin (MIC_50/90 of 0.12/0.5 mg/liter). Currently emerging high-level resistant strains (35), which represent a significant threat to the current recommended first-line dual therapy with ceftriaxone, were not included in this study but have recently been tested in another study where lefamulin showed activity (MIC_50/90 of 0.25/1 mg/ml; range, 0.004 to 2 mg/liter). Both ceftriaxone- and azithromycin-resistant strains were highly susceptible to lefamulin (36).

The development of multidrug resistance by STI pathogens is a major public health crisis, particularly for M. genitalium and N. gonorrhoeae, (4, 5, 11, 14), and new oral therapies are urgently needed. The results of the present in vitro studies showed that lefamulin was highly potent against C. trachomatis, M. genitalium, and N. gonorrhoeae and suggest that lefamulin may be a promising first-line antibiotic for the treatment of STI such as gonorrhea, nongonococcal urethritis, and cervicitis. Provided that anaerobic species are covered, lefamulin may also be a promising antibiotic for pelvic inflammatory disease, as it covers prevalent etiologies and is available in both intravenous and oral formulations. In conclusion, further studies are warranted to evaluate the efficacy and safety of lefamulin in human clinical trials of patients with STI.