Identification of bile acid and fatty acid species as candidate rapidly bactericidal agents for topical treatment of gonorrhoea

Abstract

Background

Novel therapeutic strategies are urgently needed for Neisseria gonorrhoeae, given its increasing antimicrobial resistance. Treatment of oropharyngeal N. gonorrhoeae infections has proven particularly challenging, with most reported treatment failures of the first-line drug ceftriaxone occurring at this site and lower cure rates in recent trials of new antibiotics reported for oropharyngeal infections compared with other sites of infection. However, the accessibility of the oropharynx to topical therapeutics provides an opportunity for intervention. Local delivery of a therapeutic at a high concentration would enable the use of non-traditional antimicrobial candidates, including biological molecules that exploit underlying chemical sensitivities of N. gonorrhoeae but lack the potency or pharmacokinetic profiles required for effective systemic administration.

Methods

Two classes of molecules that are thought to limit gonococcal viability in vivo, bile acids and short- and medium-chain fatty acids, were examined for rapid bactericidal activity.

Results

The bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), but not other bile acid species, exerted extremely rapid bactericidal properties against N. gonorrhoeae, reducing viability more than 100 000-fold after 1 min. The short-chain fatty acids formic acid and hexanoic acid shared this rapid bactericidal activity. All four molecules are effective against a phylogenetically diverse panel of N. gonorrhoeae strains, including clinical isolates with upregulated efflux pumps and resistance alleles to the most widely used classes of existing antimicrobials. DCA and CDCA are both approved therapeutics for non-infectious indications and are well-tolerated by cultured epithelial cells.

Conclusions

DCA and CDCA are attractive candidates for further development as anti-gonococcal agents.

Introduction

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is increasing rapidly, threatening treatment efficacy as the incidence of gonorrhoea also rises.¹ In the absence of an effective and widely available vaccine, continued control of gonorrhoea infections will require the development of novel therapeutic strategies that can surmount existing AMR.²

Most reported treatment failure of ceftriaxone, the current first-line treatment for gonorrhoea, has occurred in oropharyngeal gonorrhoea infections,^3–5 and success rates for antibiotics in recent clinical trials have been lowest for oropharyngeal compared with urogenital infections,^6–9 suggesting that this niche poses a particularly challenging environment for effective treatment.¹⁰ The extent to which this is due to unfavourable pharmacokinetics is not known, but the evidence supports the oropharynx as a critical niche for the development of AMR in N. gonorrhoeae.¹¹

In the pre-antibiotic era, topical antiseptics were a cornerstone of gonorrhoea treatment. Interest in this strategy has resurged in the face of waning efficacy of antibacterial agents.¹² Prophylactic vaginal microbicides have been proposed,¹³ although these have not thus far succeeded in clinical trials.¹⁴ A topical anti-gonorrhoeal agent may be particularly suited to the challenge of clearing oropharyngeal gonorrhoea, as it would circumvent pharmacokinetic challenges of targeting the oropharynx. Correctly applied, local treatment might also limit off-target toxicity both to the patient and to the microbiome, which in turn could reduce bystander selection for AMR in the flora.¹⁵ The development of anti-infective mouthwashes as either a prophylactic or therapeutic tool to manage oropharyngeal gonorrhoea infections has been of particular interest, with promising initial results showing reduction of viable N. gonorrhoeae loads in vitro and in patients^16,17 but no efficacy shown in clinical trials.¹⁸

One under-explored advantage of topical administration is the ability to deliver comparatively high concentrations of a drug to the site of infection, as the effective dose is not limited by systemic steady-state concentrations. This property is particularly attractive, as it reduces the potency required for an effective candidate therapeutic. As a result, almost any unusual chemical sensitivities of N. gonorrhoeae to non-toxic compounds are theoretically exploitable as a therapeutic strategy.

In N. gonorrhoeae, the MtrCDE efflux pump contributes to resistance to several antibiotic classes, including macrolides and quinolones. In natural infection, MtrCDE effluxes host-derived small hydrophobic molecules that are toxic to the bacterium. Important substrates of this pump are thought to include faecal lipids¹⁹ and bile salts^20–22 encountered during rectal colonization. The fatty acid efflux pump FarAB may also be involved in faecal lipid efflux.²³

The sensitivity of N. gonorrhoeae to these naturally occurring compounds presents a therapeutic opportunity. The promise of fatty acids as a treatment strategy has been previously noted, with particular emphasis on the sensitivity of N. gonorrhoeae to fatty acids with carbon chain lengths of ten or more.^24–26 With the exception of initial investigations into a vaginal hydrogel monocaprin prophylactic against HSV-2 in mice,²⁷ fatty acids have not been widely explored as a treatment for sexually acquired gonorrhoea infections, possibly because of their poor solubility and unfavourable pharmacokinetic properties. However, a topically applied monocaprin-based treatment for ophthalmia neonatorum is currently under development.^25,26,28,29 The promise of monocaprin as a therapeutic suggests that other topical applications of fatty acids—including for treatment of pharyngeal gonorrhoea—are a rich route for further enquiry. While the effects of medium- and long-chain fatty acids on the viability of N. gonorrhoeae have been catalogued,^24,25 there has not been a systematic investigation of short-chain fatty acids as potential gonorrhoea therapeutics.

The sensitivity of N. gonorrhoeae to different bile acid species has also not been systematically explored, although one study reported inhibitory activity of several bile acid derivatives against the laboratory strain MS11 and proposed adapting these into a prophylactic vaginal microbicide.³⁰ Larger panels of bile acids have been tested for inhibition of growth of other bacteria, such as Helicobacter pylori.³¹ Testing a larger set of bile acids for antimicrobial activity against N. gonorrhoeae may reveal additional candidates for novel topical therapeutics, especially as some bile acid species are already approved drugs for other indications (e.g. ursodeoxycholic acid for primary biliary cirrhosis; cholic acid for bile synthesis disorders; chenodeoxycholic acid for gallstone dissolution; and injectable deoxycholic acid to reduce fat below the chin).

In this work, we examine fatty acids with a range of chain lengths and a panel of bile acids for rapid bactericidal activity. Two short-chain fatty acids, formic acid and hexanoic acid, and two bile acids, deoxycholic acid and chenodeoxycholic acid, reduced viability of N. gonorrhoeae to below the limit of detection (at least 100 000-fold) after 1 min of exposure. Each of these candidates was effective at rapidly killing a range of N. gonorrhoeae strains, including clinical isolates with high-level resistance to first-line antibiotics and with hyperactive efflux pump mutations. Further development of these new candidates may enable a topical therapeutic strategy for oropharyngeal gonorrhoeae that is both rapidly effective and robust to existing AMR in the N. gonorrhoeae population.

Materials and methods

Bacterial strains and culture conditions

N. gonorrhoeae strains are presented in Table 1. All strains were cultured on GCB agar (Difco 228920) supplemented with 1% IsoVitaleX (Becton Dickinson 211876) at 37°C with 5% CO₂.

Table 1.

Neisseria gonorrhoeae strains and relevant characteristics

Strain	Antibiotic susceptibility	Relevant characteristics
FA1090	CIPS, AZIS, CROS	–
28BL	CIPS, AZIS, CROS	farA promoter variants of unknown significance: single T insertion and G > A substitution.
MS11	CIPS, AZIS, CROS	mtr 120 promoter mutation.45
H18-208	CIPR, AZIS, CRORS	Reduced cephalosporin susceptibility from penA 60.001.38
GCGS1095	CIPS, AZIS, CRORS	Reduced cephalosporin susceptibility from RpoBR201H.46
GCGS0402	CIPS, AZIR, CROS	Macrolide resistance from N. meningitides-type mosaic mtrCDE allele.36
GCGS0449	CIPS, AZIS, CROS	farA frameshift mutation (predicted FarAB loss-of-function).
GCGS0759	CIPR, AZIR, low-level CRORS	Reduced cephalosporin susceptibility from penA XXXIV;47,48 fluoroquinolone resistance from GyrA-91F/95G ParC-87R;43 increased MtrCDE expression from single A nucleotide deletion in 13-bp repeat of mtrR promoter49 likely contributing to macrolide and cephalosporin resistance; near phylogenetic neighbour of NY0195.
NY0195	CIPR, AZIS, CROS	Fluoroquinolone resistance from GyrA-91F/95G ParC-87R;43mtrC frameshift mutation (MtrCDE loss-of-function) likely contributing to phenotypic macrolide and cephalosporin susceptibility; penA XXXIV (known to confer reduced cephalosporin susceptibility);47,48 single A nucleotide deletion in 13 bp repeat of mtrR promoter (known to increase MtrCDE expression);49 near phylogenetic neighbour of GCGS0759.

The susceptibility of each strain to ciprofloxacin (CIP), azithromycin (AZI), and ceftriaxone (CRO) is presented, in addition to genotypic characteristics relevant to antimicrobial susceptibility and efflux pump activity. Susceptibility breakpoints were defined as follows: CIP^R, MIC >1 mg/L; AZI^R, MIC >2 mg/L; low-level CRO^RS, MIC 0.0625 mg/L; CRO^RS, MIC ≥0.125 mg/L. GCGS0402, GCGS0449, GCGS0759, and GCGS1095 were collected by the Centers for Disease Control and Prevention’s Gonococcal Isolate Surveillance Project.⁴³ NY0195 was collected by the New York City Public Health Laboratory, Department of Health and Mental Hygiene.⁴⁴ Sources for all strains can be found in Text S1.

Bacterial killing assays

N. gonorrhoeae strains were grown overnight on GCB agar supplemented with 1% IsoVitaleX then suspended in pre-warmed Graver-Wade medium.³² Bacterial suspensions were mixed with each bile acid or fatty acid and incubated for 60 s at ambient temperature without shaking, with a final bacterial concentration of OD₆₀₀ 0.1. Exposure was halted by immediate 10-fold dilution in Graver-Wade medium, and bacterial survival was assessed by dilution plating on GCB agar supplemented with 1% IsoVitaleX. See Text S1 for additional details (available as Supplementary data at JAC Online).

All bacterial viability assays were performed at least twice; representative data from one experiment is shown for each condition, except where repeated experiments yielded disparate results (e.g. certain short-chain fatty acids; see Supplementary data).

Cell toxicity assays

HEK 293 T and Caco-2 cells were propagated in DMEM (ATCC^® 30-2002) supplemented with 10% FBS (ATCC^® 30-2020) + 1× Penicillin-Streptomycin (Corning 30-002-CI). HeLa cells were propagated in RPMI-1640 Medium (ATCC^® 30-2001) supplemented with 10% FBS (ATCC^® 30-2020) + 1× Penicillin-Streptomycin (Corning 30-002-CI).

Cells in log phase growth were washed once with PBS without calcium and magnesium, pH 7.4 (Corning 21-040-CM), then detached with 0.25% trypsin/2.21 mM EDTA (Corning 25-053-CI) for 5–10 min and quenched in complete media. Cells were counted with an ORFLO Moxi cell counter. 50 000 cells in 0.2 mL of complete media were seeded in each well of a 96-well tissue culture plate and incubated for 18–24 h at 37°C with 5% CO₂ to allow for adherence.

Following adherence, medium was aspirated off and replaced with 0.2 mL medium supplemented with each compound or ethanol vehicle control at the specified concentration. Cells were incubated at 37°C for 60 s. Exposure was halted by removing the supplemented medium and washing three times with 0.2 mL PBS. 0.2 mL MTT reagent in medium (0.5 mg/mL) was added to each well. The contents were mixed and incubated at 37°C for 2 h. Absorbance at 540 nm (A₅₄₀) was measured on an Infinite^® M1000Pro (Tecan) reader. Cells that were subjected to media alone provided a baseline for viability at the time of assay, with percentage viability calculated as follows: [sample A₅₄₀ − blank A₅₄₀]/[baseline A₅₄₀ − blank A₅₄₀]×100. Six technical replicates were performed for each condition.

Results

Deoxycholic acid and chenodeoxycholic acid exert rapid bactericidal activity against N. gonorrhoeae

Because some bile acids are approved drugs with favourable toxicity and safety profiles, and because a topical application would allow us to deliver a high concentration to the target site, we sought to determine whether bile acids could overwhelm the MtrCDE efflux pump and cause bacterial killing.

Seven bile acids—cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, glycocholic acid, lithocholic acid, and taurocholic acid—were assayed for rapid bactericidal activity against the N. gonorrhoeae strain FA1090. Among this group, we found two bile acids that were very effective at quickly killing N. gonorrhoeae: no cfu were recovered after 60 s of exposure to either 1 mg/mL deoxycholic acid or chenodeoxycholic acid, representing a minimum of 4–5 logs of killing (Figure 1).

Figure 1.

Rapid killing of N. gonorrhoeae FA1090 by deoxycholic acid and chenodeoxycholic acid. Survival of FA1090 in Graver-Wade (GW) medium with no supplementation (-), GW containing 2% ethanol (vehicle control, V), or GW with varying concentrations of (a) deoxycholic acid or (b) chenodeoxycholic acid. Samples were mixed gently and incubated at ambient temperature for 1 min, after which exposure was halted by dilution and plating. Dotted line indicates the limit of detection (LOD, 10³ cfu/mL). Dashed line indicates bacterial survival in the vehicle control condition. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

In contrast to deoxycholic acid and chenodeoxycholic acid, none of the remaining bile acids showed bacterial killing after 60 s of exposure at a concentration of 1 mg/mL (Figure S1). This is particularly striking given the extreme similarity of the chemical structures of these bile acids. For example, no killing was observed in the presence of 1 mg/mL ursodeoxycholic acid, a stereoisomer of chenodeoxycholic acid (Figure S1D).

Initial attempts to characterize the minimal bactericidal concentration via doubling dilution found complete killing of FA1090 at 0.5 mg/mL for both deoxycholic acid and chenodeoxycholic acid, with virtually complete survival of FA1090 at 0.25 mg/mL. Dose–response experiments conducted at a finer scale showed a steep efficacy curve for both deoxycholic acid and chenodeoxycholic acid, with partial bactericidal activity between 0.3 mg/mL and 0.4 mg/mL (0.76–1.0 mM) for each of these bile acids (Figure 2). The minimal bactericidal concentration required to kill 99% of the bacterial population (MBC₉₉) was between 0.3 and 0.4 mg/mL for deoxycholic acid. The MBC₉₉ of chenodeoxycholic acid was similar, between 0.4 and 0.5 mg/mL.

Figure 2.

Dose-dependent killing of FA1090 by deoxycholic acid and chenodeoxycholic acid. Survival of FA1090 in Graver-Wade (GW) medium with no supplementation (-), GW containing 1% ethanol (vehicle control, V), or GW with varying concentrations of (a) deoxycholic acid or (b) chenodeoxycholic acid. Samples were mixed gently and incubated at ambient temperature for 1 min, after which exposure was halted by dilution and plating. Dotted line indicates the limit of detection (LOD, 10³ cfu/mL). Dashed line indicates bacterial survival in the vehicle control condition. Control groups are shown in both panels for ease of reference. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Formic acid and hexanoic acid exert rapid bactericidal activity against N. gonorrhoeae

The high sensitivity of N. gonorrhoeae to fatty acids has led to the routine inclusion of soluble starch in gonococcal growth media to sequester contaminating fatty acids³³ and is also the proposed explanation for the growth inhibition of N. gonorrhoeae by certain types of faecal lipids.¹⁹ We therefore sought to determine whether one or more fatty acids was sufficiently toxic to N. gonorrhoeae to merit consideration as a therapeutic.

A panel of fatty acids with short and medium chain lengths were tested as above for rapid bactericidal activity against the FA1090 strain. The panel comprised formic acid (C1 : 0), acetic acid (C2 : 0), propionic acid (C3 : 0), butyric acid (C4 : 0), isobutyric acid (C4 branched chain), valeric acid (C5 : 0), isovaleric acid (C5 branched chain), hexanoic acid (C6 : 0), octanoic acid (C8 : 0), decanoic acid (C10 : 0), lauric acid (C12 : 0), palmitic acid (C16 : 0), oleic acid (C18 : 1), and linoleic acid (C18 : 2). These were chosen to represent fatty acids of varying chain lengths, as well as to cover the group of faecal lipids that has previously been postulated to prevent gonococcal growth in faecal extracts (palmitic acid, oleic acid and linoleic acid).¹⁹

No viable cfu were recovered after 60 s of incubation with 1% (v/v) formic acid (C1 : 0) or hexanoic acid (C6 : 0) (Figure 3), although similar fatty acids such as acetic acid (C2 : 0), propionic acid (C3 : 0), isobutyric acid (C4), and octanoic acid (C8 : 0) failed to kill FA1090 at this timepoint, as did longer chain fatty acids (Figure S2). Several short-chain fatty acids—butyric acid (C4 : 0), valeric acid (C5 : 0), and isovaleric acid (C5) – showed promising antibacterial activity but high variability between replicates (Figure S3), which may be a result of incomplete mixing or partitioning of the fatty acid from the aqueous medium. This latter group of short-chain fatty acids may include good candidates for further testing and optimization, particularly combined with work to optimize solubility. As formic acid and hexanoic acid both resulted in consistently high bactericidal activity, we focused on further characterizing these compounds.

Figure 3.

Rapid killing of N. gonorrhoeae FA1090 by formic acid and hexanoic acid. Survival of FA1090 in Graver-Wade (GW) medium with no supplementation (-), GW containing 1% ethanol (vehicle control, V), or GW with varying concentrations of (a) formic acid or (b) hexanoic acid. Samples were mixed gently and incubated at ambient temperature for 1 min, after which exposure was halted by dilution and plating. Dotted line indicates the limit of detection (LOD, 10³ cfu/mL). Dashed line indicates bacterial survival in the vehicle control condition. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

As with deoxycholic and chenodeoxycholic acid, dose–response experiments with formic acid and hexanoic acid showed a sharp change in bactericidal activity, and doubling dilutions were not sufficient to resolve the dose–response curve. Finer-scale dose–response experiments showed partial bactericidal activity of formic acid at 0.3%–0.4% (v/v) (MBC₉₉ between 0.3% and 0.4%) (Figure 4a). Hexanoic acid was slightly less potent, with no bactericidal activity below 0.7% (v/v) (MBC₉₉ between 0.7% and 0.8%) (Figure 4b).

Figure 4.

Dose-dependent killing of FA1090 by formic acid and hexanoic acid. Survival of FA1090 in Graver-Wade (GW) medium with no supplementation (-), GW containing 1% ethanol (vehicle control, V), or GW with varying concentrations of (a) formic acid or (b) hexanoic acid. Samples were mixed gently and incubated at ambient temperature for 1 min, after which exposure was halted by dilution and plating. Dotted line indicates the limit of detection (LOD, 10³ cfu/mL). Dashed line indicates bacterial survival in the vehicle control condition. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

These compounds rapidly kill diverse clinical isolates of N. gonorrhoeae, including isolates with antimicrobial resistance

Some variants that possess resistance to existing classes of antimicrobial drugs—such as those with mutations that impact the function and regulation of the MtrCDE efflux pump resulting in increased macrolide resistance (e.g.^34–37) – could also collaterally increase resistance to fatty acids and bile salts. Because AMR in clinical N. gonorrhoeae populations is driving the need for novel therapeutics, we tested the bile acid and fatty acid candidate compounds to determine their efficacy against strains with AMR-associated alleles, including efflux pump variants.

Candidate compounds were tested against a diverse panel of N. gonorrhoeae laboratory strains and clinical isolates. The strains selected include clinically relevant resistance alleles and major variants in relevant efflux pumps, including overexpression and interspecies mosaic alleles of the MtrCDE efflux pump, as well as promoter and loss-of-function variants in the fatty acid efflux pump FarAB. These strains also included an isolate with reduced ceftriaxone susceptibility conferred by the penA 60.001 allele from the internationally disseminated FC428 clone.³⁸ Table 1 describes the strains in this panel and their relevant characteristics.

Among the eight N. gonorrhoeae strains in this panel, we saw no variability in the efficacy of rapid killing by any of the four candidate compounds: 60 s of exposure to 1 mg/mL deoxycholic acid, 1 mg/mL chenodeoxycholic acid, 1% (v/v) formic acid, or 1% (v/v) hexanoic acid in GW media was sufficient to reduce the number of viable bacteria below detectable levels in all cases (Figure 5). Resistance-associated mutations, including those thought to increase MtrCDE efflux pump activity in MS11, GCGS0759, and GCGS0402, did not reduce the efficacy of killing by either of the bile acids or the fatty acids tested.

Figure 5.

Rapid killing of diverse N. gonorrhoeae strains by deoxycholic acid, chenodeoxycholic acid, formic acid, and hexanoic acid. Survival of various strains in Graver-Wade (GW) medium without supplementation (-), GW containing ethanol (vehicle, V), or GW with (a) 1 mg/mL deoxycholic acid (yellow) or 1 mg/mL chenodeoxycholic acid (red) (vehicle control, 2% ethanol), or (b) 1% (v/v) formic acid (green) or 1% (v/v) hexanoic acid (purple) (vehicle control, 1% ethanol). Samples were mixed gently and incubated at ambient temperature for one minute, after which exposure was halted by dilution and plating. Dotted line indicates the limit of detection (LOD, 10³ cfu/mL). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Epithelial cell toxicity of candidate compounds

To evaluate the potential of each candidate molecule as a therapeutic, three epithelial cell lines were exposed to various concentrations of the candidate compounds for 60 s and cell viability was assessed by MTT assay, with a goal of determining survival of cells at concentrations at or above the MBC₉₉ of each compound for FA1090.

Formic acid did not cause significant toxicity at 0.3% (v/v), but some epithelial cell lines (Caco-2 and HEK 239 T) showed sensitivity to formic acid at concentrations above the MBC₉₉ for FA1090 (0.4% v/v and above). At 1% (v/v) formic acid, survival of all three epithelial cell lines was marginal (Figure 6a). Hexanoic acid reduced viability to below 10% for all three cell lines tested at a dose below the MBC₉₉ for FA1090 (0.7% v/v) (Figure 6b). This agrees with observations of poor viability of epidermal tissue in contact with undiluted formic or hexanoic acid after a 3 min exposure time.³⁹

Figure 6.

Epithelial cell toxicity of candidate compounds. Survival of 5 × 10⁴ adherent HeLa, Caco-2, or HEK293T cells exposed media supplemented with ethanol (vehicle, V) at 1% (panels a and b) or 2% (panels c and d), or with various concentrations of (a) formic acid, (b) hexanoic acid, (c) deoxycholic acid, or (d) chenodeoxycholic acid. After addition of each compound or vehicle control, cells were incubated at ambient temperature for 1 min, after which the medium was removed and exposure was halted by washing three times with PBS. Cell viability was evaluated by MTT assay. Shown: mean and standard deviation of six technical replicates, normalized to cell viability with no chemical or vehicle exposure (medium only). Statistical significance was tested for each condition against the appropriate vehicle control by two-tailed t-test with Bonferroni correction; *P < 0.05; **P < 0.01; ***P < 0.001. MBC₉₉ for each compound against FA1090 is shown on the x-axis for reference.

By contrast, epithelial cell viability was not compromised in the presence of up to 0.5 mg/mL deoxycholic acid or chenodeoxycholic acid, a concentration above the MBC₉₉ of these compounds for FA1090 (Figure 6c and d). Viability of Caco-2 cells in the presence of 0.3 mg/mL deoxycholic acid was significantly (but likely spuriously) increased compared with the vehicle control. Deoxycholic acid at 1 mg/mL resulted in a moderate but significant reduction in viability for all three cell lines, and 1 mg/mL chenodeoxycholic modestly reduced the viability of HEK 239 T cells.

Discussion

Oropharyngeal gonorrhoea is particularly difficult to treat, but relatively accessible to a topical therapeutic. Here, we examined two host-derived classes of small molecules that have been reported to interfere with the viability of N. gonorrhoeae. While these compounds may lack the potency to succeed as traditional systemically administered antimicrobials, using a topical formulation to deliver a locally high concentration of drug directly to the infection site could harness their rapid bactericidal activity for therapeutic use.

Bile acids are thought to be an important physiological substrate of the MtrCDE efflux pump.^19,20 Several studies have demonstrated in vitro growth inhibition of N. gonorrhoeae by cholic acid.^21,22 The absence of a functional MtrCDE pump modestly increases growth inhibition by cholic acid (roughly 2-fold decrease in MIC), as well as by glycocholic acid, taurocholic acid, and taurolithocholic acid.³⁰ We have expanded upon these observations by screening a panel of bile salts for rapid bactericidal activity, and finding that two of them—deoxycholic acid and chenodeoxycholic acid—have extremely rapid bactericidal activity, exerting at least 5 logs of bacterial killing in a 60 s exposure window. This is the first evidence that bile salts could be adapted as a rapidly effective topical therapeutic for gonorrhoea.

Some of the most promising recent work in the field of topical gonorrhoea treatment revolves around the use of monocaprin, a 10-carbon fatty acid, as a treatment for neonatal eye infection.^24–26,29 Other medium- and long-chain fatty acids also have reported in vitro antimicrobial activity against N. gonorrhoeae and are additional candidates for topical treatment.^24,25 We did not observe the large-magnitude bactericidal effects of some medium-chain fatty acids, such as lauric acid, that have been previously reported.²⁴ This may stem from differences in assay conditions, including buffer composition, drug concentration, and delivery vehicle concentration. We observed partitioning of longer-chain fatty acids in our media, which could account for variability between replicates. However, when we examined a panel of short-chain fatty acids that may be suitable for use as an oropharyngeal therapeutic, we found that formic acid and hexanoic acid are both reliably, rapidly bactericidal against N. gonorrhoeae. These compounds have not previously been reported as candidate agents for treatment of N. gonorrhoeae infection; however, the potential toxicity of these compounds may require alternative formulations. Optimizing delivery strategies and drug formulations can also minimize undesirable side effects, as in the case of a candidate bile acid therapeutic for irritable bowel syndrome.⁴⁰ This will be a critical future step in the development of these candidates for clinical use as anti-infectives.

Deoxycholic acid, chenodeoxycholic acid, formic acid, and hexanoic acid are all rapidly bactericidal against diverse strains of N. gonorrhoeae, including those with increased MtrCDE expression and high-level resistance to the antibiotics in clinical use (ceftriaxone, azithromycin, and ciprofloxacin; Table 1). It is possible that some variants represented in our strain panel might result in increased susceptibility to some of these compounds. For example, as FarAB effluxes fatty acids, the farA loss-of-function mutation in the GCGS0449 strain may increase susceptibility to formic acid, hexanoic acid, or both. As our focus was to determine whether common resistance alleles confer resistance to the candidate compounds, we did not test whether these variants reduce the MBC or increase the kinetics of bacterial death.

For each of these four candidate molecules, the mechanism of N. gonorrhoeae killing is not known. Hexanoic acid may act via a detergent-like disruption of cell membranes, which would also explain its high toxicity against epithelial cell lines. Bile salts also have detergent-like properties, but the specificity of the rapid bactericidal effect we observe—even stereospecificity, in the case of ursodeoxycholic acid versus chenodeoxycholic acid—suggests a more specific mechanism of bacterial killing. Interestingly, similar stereospecificity was observed in the growth inhibition of some Helicobacter pylori strains by chenodeoxycholic acid, but not ursodeoxycholic acid, suggesting a conserved biological distinction between these stereoisomers.⁴¹ Future work defining the mechanism of action for each of these compounds against N. gonorrhoeae will also be crucial in defining bacterial pathways to resistance, which in turn will allow us to estimate how easily resistance may arise in a clinical setting and whether implementation of fatty acid- or bile acid-based therapeutics might select for collateral resistance to other types of drugs. For example, some bile acids, including chenodeoxycholic acid, transcriptionally derepress the MtrCDE pump.⁴²

Given the need for novel antibiotics and treatment strategies, the candidate therapeutics we describe here are advantageous in many ways. First, a topical administration route such as we propose will allow us to directly target high concentrations of bactericidal compounds to the oropharynx. Second, by focusing on classes of compounds that N. gonorrhoeae is unusually sensitive to, we may be able to limit off-target effects on the normal flora, which would also help reduce bystander selection. This effect is compounded with a topical administration approach, which will limit exposure of the microbiome in other compartments, particularly in comparison to standard systemic therapy. Third, the extremely rapid bactericidal kinetics we describe here are well-suited to the challenge of treating gonorrhoea in the setting of sexual health clinics, where the simplicity of point-of-care single-dose treatment regimens (e.g. ceftriaxone) provides significant advantages. Fourth, exploring the use of small biological compounds rather than novel chemical libraries enables us to take advantage of pre-existing safety data and (in the case of some bile acids) approval for use in other clinical contexts to streamline the process of moving compounds from preclinical investigation to clinical trials. This is particularly important because there is no established animal model for pharyngeal gonorrhoea. However, in the case of candidates that are already approved therapeutics for other indications—such as deoxycholic acid and chenodeoxycholic acid—supplementing existing safety data with formulation-specific toxicity data in animals may be sufficient to permit direct progression to human trials.

The candidate compounds we describe here are well-suited to this paradigm: they are rapidly effective against diverse N. gonorrhoeae strains, including clinical isolates with substantial AMR phenotypes, and ripe for development into topical formulations.