Abstract
Secnidazole, a 5-nitroimidazole with a longer half-life, is structurally related to metronidazole and tinidazole. For treatment of bacterial vaginosis (BV), secnidazole is a suitable single-dose oral drug having a longer serum half-life than metronidazole. The objective of this study was to evaluate the antimicrobial susceptibility of vaginal isolates of facultative and anaerobic bacteria to secnidazole, metronidazole, tinidazole and clindamycin.
A total of 605 unique BV-related bacteria and 108 isolates of lactobacilli recovered from the human vagina of US women during the years 2009–2015 were tested for antimicrobial susceptibility by the agar dilution CLSI reference method to determine the minimal inhibitory concentration (MIC).
The MIC90 (μg/mL) for secnidazole was similar to metronidazole and tinidazole for Anaerococcus tetradius (secnidazole: MIC90 2; metronidazole: MIC90 2; tinidazole: MIC90 4), Atopobium vaginae (32; >128; 128), Bacteroides species (2; 2; 2), Finegoldia magna (2; 2; 4), Gardnerella vaginalis (128; 64; 32), Mageeibacillus indolicus (2; 2; 2), Megasphaera-like bacteria (0.5; 0.25; 0.5), Mobiluncus curtisii (128; >128; >128) and Mobiluncus mulieris (>128; >128; >128), Peptoniphilus lacrimalis (4; 4; 4) and Peptoniphilus harei (2; 2; 4), Porphyromonas species (0.25; 0.5; 0.25), Prevotella bivia (8; 8; 8), Prevotella amnii (2; 1; 2) and Prevotella timonensis (2; 2; 2). In this evaluation, 14 (40%) of 35 P. bivia, 5 (14%) of 35 P. amnii and 21 (58%) of 36 P. timonensis isolates were resistant to clindamycin with MIC values of >128 μg/mL. Secnidazole, like metronidazole, was superior to clindamycin for Prevotella spp., Bacteroides spp., Peptoniphilus spp., Anaerococcus tetradius and Finegoldia magna. Clindamycin had greater activity against Atopobium vaginae, Gardnerella vaginalis and Mobiluncus spp. compared to the nitroimidazoles. All 27 Lactobacillus crispatus, 26 (96%) of 27 L. jensenii, 5 (19%) of 27 L. gasseri and 18 (67%) of 27 L. iners isolates were susceptible to clindamycin (MIC ≤2) while the MIC90 for all lactobacilli tested was >128 ug/mL for secnidazole, metronidazole and tinidazole.
Secnidazole has similar in vitro activity against the range of microorganisms associated with BV compared to metronidazole or tinidazole. Further, secnidazole spares lactobacilli, a characteristic which is desirable in drugs used to treat bacterial vaginosis.
Keywords: Secnidazole, Bacterial Vaginosis, Minimal Inhibitory Concentration
Introduction
Bacterial vaginosis (BV) is a common vaginal syndrome affecting 29% of reproductive age women in the United States [1]. BV is characterized by a shift in vaginal microbiota from Lactobacillus dominance to a high diversity microbiome with increased Gardnerella vaginalis, Atopobium vaginae and other anaerobic microorganisms, such as Megasphaera, Sneathia, and Leptotrichia species [2,3]. BV has been found to be associated with an increased risk of sexually transmitted infections, including Chlamydia trachomatis [4], Neisseria gonorrhoeae [4], herpes simplex virus 1 and 2 [5,6], human immunodeficiency virus (HIV) [7,8], and Trichomonas vaginalis [4]. In addition, BV is a risk factor for reproductive health sequelae including pelvic inflammatory disease (PID) [9] and preterm birth [10].
The 2015 Centers for Disease Control and Prevention (CDC) recommended treatments for BV include oral metronidazole taken twice a day for seven days, five days of an intravaginal metronidazole gel, or seven days of an intravaginal clindamycin cream [11]. Other FDA approved treatments for BV include tinidazole taken orally for multiple days [12], a single dose metronidazole gel [13] and a single dose clindamycin cream treatment [14]. Even with a variety of antimicrobial agents available for the treatment of BV, recurrence occurs after 12 months for almost 60% of women [15]. Therefore, new therapeutic strategies are needed to more effectively treat this common condition.
Secnidazole, a second-generation 5-nitroimidazole, has a longer half-life than both metronidazole and tinidazole (~20 vs ~8 vs ~14 hours) [16] and therefore has the potential of becoming a suitable single oral dose to treat women with BV. Outside of the United States, secnidazole is used not only to treat BV but also a variety of other infections including trichomoniasis, giardiasis and amoebiasis [17, 18]. A single dose of secnidazole was shown in a European study to provide a therapeutic response similar to that of oral metronidazole twice-a-day for 7 days for treatment of BV [19]. Secnidazole, like other nitroimidazoles, also has limited activity against beneficial microbes such as Lactobacillus species, a preferred characteristic for an antibiotic used to treat vaginal infections. The objective of this study was to evaluate the antimicrobial susceptibility of vaginal isolates of facultative and anaerobic bacteria to secnidazole compared to metronidazole, clindamycin and tinidazole.
Materials and Methods
Bacterial Isolates
A total of 605 BV-associated bacteria isolates and 108 isolates of lactobacilli were recovered from the human vagina of US women during the years 2009–2015 in human subject protocols approved by the University of Pittsburgh IRB. Vaginal cultures were performed as previously described [20]. The G. vaginalis isolates were identified by their characteristic colony morphology, beta hemolysis on human bilayer agar with Tween (Becton Dickinson, Rockville, MD), Gram stain showing gram-variable pleomorphic rods, and negative catalase reaction. DNA from the anaerobic bacteria was extracted using PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA). 16S rDNA for restriction fragment length polymorphism (RFLP) analysis was completed using HaeIII (Promega, Madison, WI) restriction enzyme for identification of A. vaginae, Bacteroides species, Mageeibacillus indolicus, Mobiluncus species, and Prevotella species. Hinf1 (Promega, Madison, WI) restriction enzyme was used to identify A. tetradius, F. magna and Peptoniphilus species. Both HaeIII and Hinf1 restriction enzymes were used to identify Megasphaera-like bacteria, novel bacteria which have not yet been placed into a taxonomic group. Porphyromonas species were identified using both HaeIII and TaqI (Promega, Madison, WI) restriction enzymes. Lactobacillus species were identified using repetitive sequence polymerase chain reaction fingerprinting [21] and if needed, 16S rDNA for RFLP analysis using HpyCH4V (New England Biolab, Ipswich, MA) restriction enzymes. The RFLP patterns for each species were confirmed by 16S rDNA sequences compared to the GenBank data library using the nucleotide BLAST program.
The following organisms were included in this susceptibility study: Anaerococcus tetradius (n=30); Atopobium vaginae (n=25); Bacteroides species (n=27); Finegoldia magna (n=30); Gardnerella vaginalis (n=110); Mageeibacillus indolicus (n=11); Megasphaera-like type 1 (n=76) and type 2 (n=47); Mobiluncus curtisii (n= 51) and M. mulieris (n=12); Peptoniphilus harei (n=30) and P. lacrimalis (n=30); Porphyromonas species (n=20); Prevotella amnii (n=35), P. bivia (n=35) and P. timonensis (n=36); and Lactobacillus crispatus (n=27), L. gasseri (n=27), L. iners (n=27) and L. jensenii (n=27).
Agar Dilution Susceptibility Testing
The vaginal isolates were evaluated for susceptibility to secnidazole (Symbiomix, Newark, NJ), metronidazole, tinidazole and clindamycin (all from Sigma-Aldridge, St. Louis, MO) using the anaerobic agar dilution method described by the Clinical and Laboratory Standards Institute [22,23].
The concentrations of antimicrobial agents used ranged from 0.03 to 128 μg/mL. Prior to testing, Lactobacillus species were cultivated on Columbia agar with 5% sheep blood (Becton Dickinson, Rockville, MD) and incubated in anaerobic jars, G. vaginalis isolates were cultivated anaerobically on human bilayer agar with Tween (Becton Dickinson, Rockville, MD), and all other isolates were grown anaerobically on Brucella agar (Hardy Diagnostics, Santa Maria, CA). The isolates were isolated to purity and suspended in Brucella broth (Becton Dickinson, Rockville, MD) at a 0.5 McFarland suspension. Using a Steer’s replicator, Brucella agar (Remel, Lenexa, KS) plates with 5% Laked Sheep Blood (Hardy Diagnostic, Santa Maria, CA) and varying concentrations of test agent alongside a no drug growth control were inoculated and incubated in anaerobic jars for 48 hours at 37°C. The lowest antibiotic concentration yielding marked reduction to no growth was read as the Minimum Inhibitory Concentration (MIC). Three control strains, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium difficile ATCC 700057 (American Type Culture Collection, Rockville, MD) were used to ensure quality of testing. G. vaginalis ATCC 14018 was also used as a supplemental control only when testing G. vaginalis isolates. The microbiological susceptibility and resistant breakpoints for clindamycin (≤2μg/mL and ≥8μg/mL) and metronidazole (≤8μg/mL and ≥32μg/mL) as defined by CLSI [22, 23] were used for interpretation of MIC results. CLSI does not have a defined susceptibility or resistant breakpoint for either secnidazole or tinidazole.
Results
The 713 female genital tract isolates were evaluated for susceptibility to secnidazole, clindamycin, metronidazole and tinidazole. As summarized in Tables 1–4, MIC50 and MIC90 values were similar for secnidazole, metronidazole and tinidazole for Anaerococcus tetradius, A. vaginae, Bacteroides species, Finegoldia magna, G. vaginalis, Mageeibacillus indolicus, Megasphaera-like bacteria, Mobiluncus curtisii and M. mulieris, Peptoniphilus harei and P. lacrimalis, Porphyromonas species, Prevotella bivia, P. amnii and P. timonensis, and all four Lactobacillus species.
Table 1.
The comparative in vitro activity of secnidazole, metronidazole, tinidazole and clindamycin against 253 bacterial vaginosis (BV) related organisms susceptible to metronidazole
| MIC (ug/mL) | ||||||
|---|---|---|---|---|---|---|
| Species | Na | Antimicrobial Agent | Range | 50% | 90% | nb (%)c |
| Anaerococcus tetradius | 30 | Clindamycin | 0.5 – >128 | 1 | >128 | 6 (20) |
| Metronidazole | 1 – 4 | 2 | 2 | 0 | ||
| Secnidazole | 0.5 – 4 | 2 | 2 | NDd | ||
| Tinidazole | 0.5 – 4 | 2 | 4 | ND | ||
| Bacteroides speciese | 27 | Clindamycin | 0.03 – >128 | 1 | >128 | 8 (30) |
| Metronidazole | 0.25 – 2 | 1 | 2 | 0 | ||
| Secnidazole | 0.5 – 4 | 1 | 2 | ND | ||
| Tinidazole | 0.25 – 2 | 1 | 2 | ND | ||
| Finegoldia magna | 30 | Clindamycin | 0.5 – >128 | 2 | >128 | 10 (33) |
| Metronidazole | 0.5 – 8 | 1 | 2 | 0 | ||
| Secnidazole | 0.5 – 8 | 2 | 2 | ND | ||
| Tinidazole | 0.5 – 16 | 2 | 4 | ND | ||
| Peptoniphilus harei | 30 | Clindamycin | 3.125 – >128 | 0.5 | 8 | 4 (13) |
| Metronidazole | 1 – 4 | 2 | 2 | 0 | ||
| Secnidazole | 1 – 4 | 2 | 2 | ND | ||
| Tinidazole | 2 – 4 | 2 | 4 | ND | ||
| Peptoniphilus lacrimalis | 30 | Clindamycin | 3.125 – >128 | 64 | >128 | 19 (63) |
| Metronidazole | 0.125 – 4 | 1 | 4 | 0 | ||
| Secnidazole | 0.25 – 4 | 2 | 4 | ND | ||
| Tinidazole | 0.125 – 4 | 2 | 4 | ND | ||
| Prevotella amnii | 35 | Clindamycin | 0.03 – >128 | 0.03 | >128 | 5 (14) |
| Metronidazole | 0.25 – 4 | 0.5 | 1 | 0 | ||
| Secnidazole | 0.25 – 4 | 1 | 2 | ND | ||
| Tinidazole | 0.5 – 2 | 1 | 2 | ND | ||
| Prevotella bivia | 35 | Clindamycin | 0.03 – >128 | 0.06 | >128 | 14 (40) |
| Metronidazole | 1 – 8 | 4 | 8 | 0 | ||
| Secnidazole | 0.5 – 16 | 4 | 8 | ND | ||
| Tinidazole | 2 – 8 | 4 | 8 | ND | ||
| Prevotella timonensis | 36 | Clindamycin | 0.03 – >128 | >128 | >128 | 21 (58) |
| Metronidazole | 0.25 – 8 | 2 | 2 | 0 | ||
| Secnidazole | 0.5 – 4 | 2 | 2 | ND | ||
| Tinidazole | 0.125 – 2 | 1 | 2 | ND | ||
Number of isolates tested
Number of resistant isolates
Percent resistant (n/N)
Resistance is not defined (ND) for secnidazole and tinidazole by CLSI
Bacteroides species includes 2 B. caccae, 1 B. clarus, 1 B. fragilis, 4 B. ovatus, 2 B. salyersiae, 1 B. species, 2 B. stercoris, 1 B. thetaaitaomicron, 3 B. uniformis, 6 B. vulgatus, 2 B. vulgatus/dorei and 2 B. xylanisolvens
Table 4.
The comparative in vitro activity of secnidazole, metronidazole, tinidazole and clindamycin against 108 Lactobacillus species
| MIC (ug/mL) | ||||||
|---|---|---|---|---|---|---|
| Species | Na | Antimicrobial Agent | Range | 50% | 90% | nb (%)c |
| Lactobacillus crispatus | 27 | Clindamycin | 0.03 – 0.5 | 0.125 | 0.125 | 0 |
| Metronidazole | >128 | >128 | >128 | 27 (100) | ||
| Secnidazole | 1 – >128 | >128 | >128 | NDd | ||
| Tinidazole | 0.06 – >128 | 16 | >128 | ND | ||
| Lactobacillus gasseri | 27 | Clindamycin | 0.25 – 8 | 4 | 8 | 12 (44) |
| Metronidazole | >128 | >128 | >128 | 27 (100) | ||
| Secnidazole | >128 | >128 | >128 | ND | ||
| Tinidazole | >128 | >128 | >128 | ND | ||
| Lactobacillus iners | 27 | Clindamycin | 0.125 – >128 | 0.5 | >128 | 9 (33) |
| Metronidazole | >128 | >128 | >128 | 27 (100) | ||
| Secnidazole | >128 | >128 | >128 | ND | ||
| Tinidazole | >128 | >128 | >128 | ND | ||
| Lactobacillus jensenii | 27 | Clindamycin | 0.25 – 8 | 0.5 | 1 | 1 (4) |
| Metronidazole | >128 | >128 | >128 | 27 (100) | ||
| Secnidazole | >128 | >128 | >128 | ND | ||
| Tinidazole | >128 | >128 | >128 | ND | ||
Number of isolates tested
Number of resistant isolates
Percent resistant (n/N)
Resistance is not defined (ND) for secnidazole and tinidazole by CLSI
BV-associated bacteria had susceptibility patterns for clindamycin and the three nitroimidazoles that distributed into three distinct groups. The first group of BV-associated bacteria (Table 1) consisted of anaerobic gram-negative rods and anaerobic gram-positive cocci which were susceptible to metronidazole based on a CLSI susceptibility breakpoint of ≤8 μg/mL. For secnidazole, only two P. bivia isolates had a MIC value of 16 μg/mL. One F. magna isolate also had a MIC of 16 μg/mL for tinidazole. All of the other isolates had MIC values ≤8 μg/mL for secnidazole and tinidazole. Clindamycin resistance, on the other hand, was observed in 38% of Prevotella species, 30% of Bacteroides species, 38% of Peptoniphilus species, 20% of Anaerococcus tetradius isolates, and 33% of Finegoldia magna isolates tested. All of the Prevotella and Bacteroides species resistant to clindamycin had MIC values of >128 μg/mL.
A second group of BV-associated bacteria (Table 2) consisted of anaerobic gram-positive and facultative gram-variable rods that were susceptible to clindamycin but had varying degrees of resistance to metronidazole. These included 9 M. curtisii isolates resistant to clindamycin (MIC values ≥8 μg/mL) and half of the A. vaginalis, G. vaginalis and Mobiluncus isolates having metronidazole resistance (MIC values ≥32 μg/mL). Secnidazole had a very similar range of activity against these BV-associated bacteria compared to metronidazole. Overall, 64% A. vaginae, 30% G. vaginalis, 69% M. curtisii and 42% M. mulieris isolates had secnidazole MIC values ≥32 μg/mL. Likewise, tinidazole had MIC values of ≥32 μg/mL for 72% A. vaginae, 22% G. vaginalis, 82% M. curtisii and 42% M. mulieris isolates. Similar rates of resistance were observed for metronidazole among these species.
Table 2.
The comparative in vitro activity of secnidazole, metronidazole, tinidazole and clindamycin against 198 bacterial vaginosis (BV) related organisms susceptible to clindamycin
| MIC (ug/mL) | ||||||
|---|---|---|---|---|---|---|
| Species | Na | Antimicrobial Agent | Range | 50% | 90% | nb (%)c |
| Atopobium vaginae | 25 | Clindamycin | 0.03 – 0.125 | 0.125 | 0.125 | 0 |
| Metronidazole | 8 – >128 | 64 | >128 | 18 (72) | ||
| Secnidazole | 8 – 64 | 32 | 32 | NDd | ||
| Tinidazole | 0.25 – >128 | 32 | 128 | ND | ||
| Gardnerella vaginalis | 110 | Clindamycin | 0.03 – 0.5 | 0.125 | 0.5 | 0 |
| Metronidazole | 1 – >128 | 8 | 64 | 30 (27) | ||
| Secnidazole | 1 – >128 | 8 | 128 | ND | ||
| Tinidazole | 0.25 – >128 | 4 | 32 | ND | ||
| Mobiluncus curtisii | 51 | Clindamycin | 0.06 – >128 | 0.125 | >128 | 9 (18) |
| Metronidazole | 2 – >128 | 128 | >128 | 46 (90) | ||
| Secnidazole | 1 – >128 | 128 | 128 | ND | ||
| Tinidazole | 2 – >128 | >128 | >128 | ND | ||
| Mobiluncus mulieris | 12 | Clindamycin | 0.03 – 0.25 | 0.06 | 0.125 | 0 |
| Metronidazole | 2 – >128 | 8 | >128 | 5 (42) | ||
| Secnidazole | 4 – >128 | 8 | >128 | ND | ||
| Tinidazole | 2 – >128 | 8 | >128 | ND | ||
Number of isolates tested
Number of resistant isolates
Percent resistant (n/N)
Resistance is not defined (ND) for secnidazole and tinidazole by CLSI
A third group of BV-associated bacteria (Table 3) were susceptible to both clindamycin and metronidazole including the novel bacteria Mageeibacillus indolicus and Megasphaera-like type I and II bacteria. For secnidazole and tinidazole, the highest MIC concentration among Megasphaera-like bacteria isolates was 1 μg/mL with the exception of one Megasphaera-like type I isolate which had an MIC value of 16 μg/mL. All of the Mageeibacillus indolicus and Porphyromonas species isolates had MIC values of ≤2 μg/mL for secnidazole and tinidazole. Only one Porphyromonas asaccharolytica isolate was resistant to clindamycin.
Table 3.
The comparative in vitro activity of secnidazole, metronidazole, tinidazole and clindamycin against 154 bacterial vaginosis (BV) related organisms susceptible to clindamycin and metronidazole
| MIC (ug/mL) | ||||||
|---|---|---|---|---|---|---|
| Species | Na | Antimicrobial Agent | Range | 50% | 90% | nb (%)c |
| Mageeibacillus indolicus (BVAB3) | 11 | Clindamycin | 0.06 – 1 | 0.06 | 1 | 0 |
| Metronidazole | 1 – 2 | 2 | 2 | 0 | ||
| Secnidazole | 1 – 2 | 1 | 2 | NDd | ||
| Tinidazole | 0.5 – 2 | 0.5 | 2 | ND | ||
| Megasphaera-like type I | 76 | Clindamycin | 0.03–2 | 0.03 | 0.06 | 0 |
| Metronidazole | 0.03–8 | 0.25 | 0.25 | 0 | ||
| Secnidazole | 0.03–16 | 0.5 | 1 | ND | ||
| Tinidazole | 0.03–16 | 0.5 | 0.5 | ND | ||
| Megasphaera-like type II | 47 | Clindamycin | 0.03–2 | 0.03 | 0.125 | 0 |
| Metronidazole | 0.03–0.5 | 0.125 | 0.25 | 0 | ||
| Secnidazole | 0.03–1 | 0.25 | 0.5 | ND | ||
| Tinidazole | 0.03–1 | 0.25 | 0.5 | ND | ||
| Porphyromonas speciese | 20 | Clindamycin | 0.03 – 64 | 0.03 | 0.125 | 1 (5) |
| Metronidazole | 0.06 – 1 | 0.25 | 0.5 | 0 | ||
| Secnidazole | 0.03 – 2 | 0.125 | 0.25 | ND | ||
| Tinidazole | 0.03 – 2 | 0.125 | 0.25 | ND | ||
Number of isolates tested
Number of resistant isolates
Percent resistant (n/N)
Resistance is not defined (ND) for secnidazole and tinidazole by CLSI
Porphyromonas species includes 10 P. asaccharolytica and 10 P. uenonis
As summarized in Table 4, metronidazole and tinidazole had no activity against any of the lactobacilli tested. Secnidazole had no activity (MICs >128μg/mL) against L. crispatus (excluding one isolate with an MIC of 1 μg/mL, 2 with an MIC of 8 μg/mL, and one with an MIC of 64 μg/mL), L. gasseri, L. iners or L. jensenii. All 27 L. crispatus, 96% L. jensenii, 19% L. gasseri and 67% L. iners were susceptible to clindamycin (MIC ≤2).
Discussion
The present study evaluated the susceptibility of a broad range of vaginal isolates to four antibiotics used to treat bacterial vaginosis. The taxonomic status of many of the microorganisms associated with bacterial vaginosis has changed in the past several years making it difficult to compare the present results to those from previously published studies for individual microorganisms. Clindamycin resistance among anaerobic isolates recovered from women with bacterial vaginosis has been reported previously [24]. The data generated in this study suggests that greater than a third of Prevotella species colonizing the vagina may be resistant to clindamycin.
Metronidazole remains the most commonly used antimicrobial agent for the treatment of BV despite limited in vitro activity against G. vaginalis and A. vaginae, both of which are uniformly present among women with BV. Secnidazole had a very similar range of activity in vitro against these microorganisms compared to metronidazole. Megasphaera-like bacteria have been described as being strongly associated with BV using culture independent methods and were until recently considered to be noncultivable. The data generated from this study suggests that this novel Gram-negative microorganism is susceptible to both nitroimidazoles and clindamycin. Consistent with previously published data [25], the nitroimidazoles provide excellent coverage against most of the anaerobic microbiota associated with bacterial vaginosis. Nitroimidazoles have no in vitro activity against lactobacilli recovered from the vagina [26] while clindamycin has activity against these beneficial microorganisms as shown in the present study.
In summary, the data generated from these studies suggests that secnidazole has similar activity against the range of microorganisms associated with BV compared to metronidazole or tinidazole. Further, secnidazole spares lactobacilli, a characteristic which is desirable in drugs used to treat bacterial vaginosis. Taken together, these results illustrate the potential of secnidazole for the treatment of bacterial vaginosis.
Highlights.
Secnidazole, an azole drug with a longer half-life than metronidazole, has activity against the bacteria associated with bacterial vaginosis.
Like metronidazole, secnidazole has limited activity against vaginal Lactobacillus species.
Secnidazole has the potential to be used for treatment of bacterial vaginosis.
Acknowledgments
This work was supported by Symbiomix Therapeutics, LLC, Baltimore, MD and the following grants from the National Institutes of Health: AI082639, AI102835 and AI084024.
Footnotes
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