Antimicrobial effects of Medicines for Malaria Venture Pathogen Box compounds on strains of Neisseria gonorrhoeae

Eric Mensah; P Bernard Fourie; Remco P H Peters

doi:10.1128/aac.00348-23

. 2023 Oct 4;67(11):e00348-23. doi: 10.1128/aac.00348-23

Antimicrobial effects of Medicines for Malaria Venture Pathogen Box compounds on strains of Neisseria gonorrhoeae

Eric Mensah ¹, P Bernard Fourie ¹, Remco P H Peters ^1,^2,^✉

Editor: Audrey Odom John³

PMCID: PMC10648949 PMID: 37791750

ABSTRACT

Therapeutic options for Neisseria gonorrhoeae are limited due to emerging global resistance. New agents and treatment options to treat patients with susceptible and multi-extensively drug-resistant N. gonorrhoeae is a high priority. This study used an in vitro approach to explore the antimicrobial potential, as well as synergistic effects of Medicine for Malaria Venture (MMV) Pathogen Box compounds against ATCC and clinical N. gonorrhoeae strains. Microbroth dilution assay was used to determine pathogen-specific minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Pathogen Box compounds against susceptible and resistant N. gonorrhoeae strains, with modification, by adding PrestoBlue HS Cell Viability Reagent. A checkerboard assay was used to determine synergy between the active compounds and in conjunction with ceftriaxone. Time-kill kinetics was performed to determine if the compounds were either bactericidal or bacteriostatic. The Pathogen Box compounds: MMV676501, MMV002817, MMV688327, MMV688508, MMV024937, MMV687798 (levofloxacin), MMV021013, and MMV688978 (auranofin) showed potent activity against resistant strains of N. gonorrhoeae at an MIC and MBC of ≤10 µM. Besides the eight compounds, MMV676388 and MMV272144 were active against susceptible N. gonorrhoeae strains, also at MIC and MBC of ≤10 µM. All the compounds were bactericidal and were either synergistic or additive with fractional inhibitory concentration index ranging between 0.40 and 1.8. The study identified novel Pathogen Box compounds with potent activity against N. gonorrhoeae strains and has the potential to be further investigated as primary or adjunctive therapy to treat gonococcal infections.

KEYWORDS: Neisseria gonorrhoeae, novel compound, drug discovery, MMV Pathogen Box, in vitro bactericidal activity

INTRODUCTION

Gonococcal infections caused by Neisseria gonorrhoeae (N. gonorrhoeae) are important sexually transmitted infections globally (1, 2). According to the World Health Organization (WHO), approximately 106 million new cases of gonococcal infections occur annually worldwide (3). Increasing evidence suggests that the actual number of these infections is under-reported because of inadequate clinical or diagnostic infrastructure, poor reporting systems, and high rates of asymptomatic infections (4 – 7). Without effective treatment, these infections can result in severe complications such as pelvic inflammatory disease, increased risk of tubal factor infertility, ectopic pregnancy and adverse pregnancy outcomes, and facilitate the transmission of the human immunodeficiency virus (HIV) (8 – 10).

The emergence of N. gonorrhoeae strains resistant to all drugs recommended for treatment, including third-generation cephalosporins, azithromycin, fluoroquinolones, tetracyclines, and β-lactams, is a major public health concern (3, 11 – 13). The WHO has designated fluoroquinolone and third-generation cephalosporin-resistant N. gonorrhoeae as a high-priority pathogen (14). Dual therapy with ceftriaxone and azithromycin has been the mainstay for the treatment of gonococcal infections for the past decade (15 – 18). However, because of the potent anti-commensal activity of the dual therapy and increasing resistance to azithromycin, the USA and UK have removed azithromycin from the treatment regimen; ceftriaxone monotherapy is now recommended for treating N. gonorrhoeae infections (19). Emergence of infections with resistance to ceftriaxone is reported in different countries, making N. gonorrhoeae a superbug, requiring urgent development of new drugs and therapeutic options (10, 20 – 26). The continuous increase in antibiotic resistance, coupled with the limited pipeline and availability of new drugs has raised global concern about the emergence of untreatable gonorrhoea. No currently available vaccine to prevent infections highlights the critical need to develop new treatment agents, even for susceptible N. gonorrhoeae since ceftriaxone is the only treatment option currently.

In order to accelerate the discovery of novel drug compounds, the Medicines for Malaria Venture (MMV) group has developed the Pathogen Box, a collection of 400 drugs that have demonstrated biological activity against specific pathogenic organisms in a screen that was initially mostly directed at protozoal parasites responsible for tropical diseases, in particular malaria (27). These 400 compounds are mostly novel synthetic chemicals that were initially selected from a screen of over 4 million chemicals due to their low toxicity to mammalian cells. Each of the compounds has been tested for cytotoxicity and has shown values within levels considered acceptable for an initial drug discovery program (28). The activity of the Pathogen Box compounds against some bacteria including Acinetobacter baumannii (29) and Staphylococcus aureus (30) has been reported. However, activity against resistant forms of several key pathogens, including N. gonorrhoeae, has not been explored. In this study, we conducted in vitro testing of the antibiotic potential, including synergistic effects of MMV Pathogen Box compounds against N. gonorrhoeae strains.

RESULTS

Testing for antimicrobial activity of Pathogen Box compounds against N. gonorrhoeae strains

Table 1 shows the pathogen-specific minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Pathogen Box compounds against N. gonorrhoeae strains using broth microdilution assay. All the 400 drug molecules, including reference drugs, were screened against N. gonorrhoeae isolates at 10 µM. After the primary screen, 25 compounds showed partial or full inhibitory effect against N. gonorrhoeae isolates at 10 µM (Fig. S1). Since the MIC of the most effective drug, ceftriaxone, is ≤0.189 µM (≤ 0.125 µg/mL) (31), we used concentrations ranging between 0.156 and 10 µM to identify compounds with full activity and determine the pathogen-specific MIC and MBC using a twofold serial dilution broth microdilution (Fig. S2). The activity of these compounds was repeated in triplicate. Eight compounds, MMV676501 (Pbc 1), MMV002817 (Pbc 3), MMV688327 (Pbc 4, Radezolid), MMV688508 (Pbc 5), MMV024937 (Pbc 6), MMV687798 (Pbc 7, levofloxacin), MMV021013 (Pbc 9), and MMV688978 (Pbc 10, auranofin), showed full activity against the N. gonorrhoeae ATCC (Fig. S3C) and clinical strains (Fig. S3A and B). In addition, MMV676388 (Pbc 2) and MMV272144 (Pbc 8) showed full activity against the N. gonorrhoeae ATCC 49266 strain but only partial activity against the clinical strains. Among the 10 drug compounds, two were reference drugs: MMV687798 (Pbc 7, levofloxacin) and MMV688978 (Pbc 10, auranofin). The remaining eight were novel compounds with potent activity against N. gonorrhoeae strains. The MIC and MBC were much lower in the susceptible ATCC N. gonorrhoeae strains than in the clinical strains in most of the compounds (Table 1). The MIC of MMV676501 (Pbc 1) was 0.625 µM for all the isolates irrespective of the resistance profile. The MIC and MBC of MMV687798 (Pbc 7, Levofloxacin) were <0.0195 µM in reference strain and 5 µM in clinical strain 1 (Ciprofloxacin MIC = 0.5 mg/L) and 10 µM in clinical strain 2 (Ciprofloxacin MIC = 2 mg/L). The MIC of MMV688978 (Pbc 10, Auranofin) (0.3125 µM), MMV002817 (Pbc 3) (2.5 µM), MMV688327 (Pbc 4, Radezolid) (2.5 µM), MMV024937 (Pbc 6) (10 µM), and MMV021013 (Pbc 9) (5 µM) was the same in both clinical strain 1 and clinical strain 2 (Table 1).

TABLE 2.

Effect of combination of four best Pathogen Box compounds ^a

Test compounds	MIC (µM)		FICI	Interpretation
Test compounds	MIC alone	MIC in combination		SYN	ADD	IND
MMV676501 (Pbc 1)	0.625	0.5	1.3	IND
MMV024937 (Pbc 6)	10	5	1.3	IND
MMV676501 (Pbc 1)	0.625	0.5	1.8	IND
MMV687798 (Pbc 7, levofloxacin)	5	5	1.8	IND
MMV676501 (Pbc 1)	0.625	0.5	1.6	IND
MMV688978 (Pbc 10, auranofin)	0.3125	0.25	1.6	IND
MMV024937 (Pbc 6)	10	2.5	0.75	ADD
MMV687798 (Pbc 7, levofloxacin)	5	2.5	0.75	ADD
MMV024937 (Pbc 6)	10	2.5	1.05	ADD
MMV688978 (Pbc 10, auranofin)	0.3125	0.25	1.05	ADD
MMV687798 (Pbc 7, levofloxacin)	5	2.5	1.3	IND
MMV688978 (Pbc 10, auranofin)	0.3125	0.25	1.3	IND

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^{^a}

FICI, fractional inhibitory concentration index; Add, additive; Syn, synergistic; IND, indifference; A FICI value of ≤0.5 was considered a synergistic activity; between >0.5 but ≤1.25 as an additive activity; >1.25 − ≤4 as indifference. The four best compounds, MMV676501, MMV024937, MMV687798, and MMV688978, were combined at a concentration lower than their respective MIC.

TABLE 1.

Pathogen-specific MIC and MBC of Pathogen Box compounds against susceptible and resistant N. gonorrhoeae strains ^a

MMV ID	Compound class (common name)	Molecular formula/structure ^b	Pathogen Box target ^b	Mode of action	HepG2 CC₂₀ (μM) ^b	ATCC 49226		Clinical strain 1		Clinical strain 2
MMV ID	Compound class (common name)	Molecular formula/structure ^b	Pathogen Box target ^b	Mode of action	HepG2 CC₂₀ (μM) ^b	MIC (µM)	MBC (µM)	MIC (µM)	MBC (µM)	MIC (µM)	MBC (µM)
MMV676501 (Pbc 1)	-	C₁₁H₅N₃O₂Cl₂S₂	Mycobacterium tuberculosis	Unknown	2.64	0.625	0.625	0.625	1.25	0.625	1.25
MMV676388 (Pbc 2)	5-Sulfonyl tetrazole	C₁₅H₁₄N₄O₃S	M. tuberculosis	Unknown	1.85	5	5	ǂ	ǂ	ǂ	ǂ
MMV002817 (Pbc 3)	Diiodohydroxyquinoline (iodoquinol)	C₉H₅NOI₂	Lymphatic filariasis-onchocerciasis	Chelates ferrous ions required for amoebic metabolism	2.53	1.25	1.25	2.5	2.5	2.5	2.5
MMV688327 (Pbc 4)	Oxazolidinone (radezolid)	C₂₂H₂₃N₆O₃F	M. tuberculosis	Inhibit 50S ribosomal subunit during protein synthesis	8	0.3125	0.3125	2.5	2.5	2.5	2.5
MMV688508 (Pbc 5)	Oxazolidinone	C₁₉H₁₉N₂O₄F	M. tuberculosis	Inhibit 50S ribosomal subunit during protein synthesis	5.87	0.625	0.625	2.5	5	2.5	5
MMV024937 (Pbc 6)	– ^c	C₂₀H₁₈N₅O₂F₃	Plasmodium falciparium	Unknown	13.4	5	5	10	10	10	10
MMV687798 (Pbc 7)	Quinolone (levofloxacin)	C₁₈H₂₀N₃O₄F	Broad range spectrum	Inhibit bacterial DNA gyrase and topoisomerase IV.	>80	≤0.0195	≤0.0195	5	5	10	10
MMV272144 (Pbc 8)	Heteroaromatic sulfones	C₉H₁₀N₄O₃S	M. tuberculosis	Unknown	0.764	10	10	ǂ	ǂ	ǂ	ǂ
MMV021013 (Pbc 9)	2-Pyridyl-4-aminopyrimidine	C₁₈H₂₂N₄	M. tuberculosis	Unknown	0.629	1.25	1.25	5	5	5	5
MMV688978 (Pbc 10)	(Auranofin)	C₂₀H₃₄AuO₉PS	Rheumatoid arthritis drug recently repurposed against amebiasis	Inhibits bacterial thioredoxin reductase	1.74	0.156	0.156	0.3125	0.3125	0.3125	0.3125

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^{^a}

Profile of 10 Pathogen Box compounds with potent activity against N. gonorrhoeae strains at ≤10 µM. ǂ, partial activity at 10 µM; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration. NA, not applicable.

^{^b}

Information provided by Medicines for Malaria Venture.

^{^c}

– Unnamed compound.

Time-kill kinetics of Pathogen Box against N. gonorrhoeae

After confirming the antibacterial activity of eight Pathogen Box compounds against two N. gonorrhoeae clinical strains (Table 1) using microbroth dilution, we examined whether these molecules (all at 3× MIC) exhibit bacteriostatic or bactericidal activity via standard time-kill kinetic assay over 24 hours. As presented in Fig. 1, all eight compounds showed bactericidal activity against clinical strain 1, which was chosen because of the highest MIC of azithromycin. After 4 hours, auranofin exhibited rapid bactericidal activity. Additionally, the time-kill curve of the combination of the four best compounds, and in conjunction with ceftriaxone, found to be either synergistic or additive was also plotted (Tables 2 and 3; Fig. 2). The dual combinations demonstrated bactericidal activity over 24 hours. The compounds showed similar time-kill activity to ceftriaxone in our experimental design (Fig. 1 and 2). After 6 hours, the combination of Pbc 6 and Pbc 10 resulted in complete bacteriocidal activity (Fig. 2).

Fig 1 — Time-kill analysis of Pathogen Box compounds (at 3× MIC) against *N. gonorrhoeae* over a 24-hour incubation period at 35ºC. DMSO served as a negative control.

TABLE 3.

Effect of combination of ceftriaxone with four best compounds ^a

Test compounds	MIC (µM)		FICI	Interpretation
Test compounds	MIC alone	MIC in combination		SYN	ADD	IND
Ceftriaxone	0.00244	0.0015625	1.44	IND
MMV676501 (Pbc 1)	0.625	0.5	1.44	IND
Ceftriaxone	0.00244	0.0015625	0.84	ADD
MMV024937 (Pbc 6)	10	2	0.84	ADD
Ceftriaxone	0.00244	0.0015625	1.04	ADD
MMV687798 (Pbc 7, levofloxacin)	5	2	1.04	ADD
Ceftriaxone	0.00244	0.00078125	1.12	ADD
MMV688978 (Pbc 10, auranofin)	0.3125	0.25	1.12	ADD

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^{^a}

TABLE 4.

Antimicrobial susceptibility profile of N. gonorrhoeae ATCC 49266 and clinical strains ^a

Isolate (accession number)	MIC (mg/L) of antibiotics						Resistance-associated mutations
	Azithromycin (0.016–256) ^b	Ceftriaxone (0.016–256) ^b	Cefixime (0.016–256) ^b	Ciprofloxacin (0.002–32) ^b	Tetracycline (0.016–256) ^b	Penicillin (0.016–256) ^b
ATCC AT49226	0.032 (S)	<0.002 (S)	<0.016 (S)	<0.002 (S)	0.047(S)	<0.016 (S)	–
Clinical strain 1 (SRS5471848)	0.75 (S)	<0.002 (S)	<0.016 (S)	0.5 (R)	12 (R)	0.38 (R)	GyrA, tetM, bla _TEM
Clinical strain 2 (SRS5471840)	0.38 (S)	0.002 (S)	<0.016 (S)	2 (R)	12 (R)	3 (R)	GyrA, tetM, bla _TEM

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^{^a}

Antimicrobial susceptibility profile of N. gonorrhoeae ATCC 49266 and clinical strains isolated from symptomatic male patients. S, susceptible; R, resistant.

^{^b}

Range of antibiotics tested.

^{^c}

– Wild-type; non-identified.

Fig 2 — Time-kill kinetics of the combination of four best compounds, and in conjunction with ceftriaxone, found to be additive (at 3× MIC in combination) against *N. gonorrhoeae* over a 24-hour incubation period at 35°C. DMSO served as a negative control.

Combination testing of Pathogen Box compounds and in conjunction with ceftriaxone against N. gonorrhoeae

We investigated the combination of eight Pathogen Box compounds and in conjunction with ceftriaxone. Ceftriaxone was selected because it is the drug of choice for treating N. gonorrhoea. As presented in Tables 2 and 3 and Tables S1 to S7, the compounds show synergistic and additive activity against N. gonorrhoeae isolates. Fractional inhibitory concentration indices (FICIs) ranged from 0.45 to 1.8. Four best Pathogen Box compounds, Pbc 1, Pbc 6, Pbc 7, and Pbc 10, were selected and combined with themselves and in conjunction with ceftriaxone (Tables 2 and 3). There was an additive effect between ceftriaxone, MMV024937 (Pbc 6), MMV687798 (Pbc 7, levofloxacin), and MMV688978 (Pbc 10, auranofin) (Table 3).

DISCUSSION

Neisseria gonorrhoeae is one of the high-priority pathogens defined by the World Health Organization because of its exceptional capacity to acquire resistance to all antimicrobials used as first-line drugs for treatment (10, 14, 25). Even though N. gonorrhoeae continues to evolve resistance rapidly, the rate at which new antibiotics are discovered and developed has steadily decreased (32, 33). The emergence of multidrug-resistant (MDR) and extensively drug-resistant strains has worsened the situation. The development of high-level resistance to last resort ceftriaxone, leading to untreatable gonorrhoea, coupled with the limited pipeline of new anti-gonococcal drugs highlights the urgent need to discover new antibacterial agents (6, 21, 33, 34). We screened the MMV Pathogen Box that has previously demonstrated biological activity against specific pathogenic organisms to identify novel treatment options or drug molecules with activity against N. gonorrhoeae using an in vitro approach instead of in silico or mechanistic approach (35 – 37). We identified 10 Pathogen Box compounds with potent activity against N. gonorrhoeae strains, with the potential for further research. Two of the compounds are reference drugs, auranofin, and levofloxacin while the other eight are novel compounds.

Broth microdilution assay, a non-standard method, was used to explore antimicrobial activity due to the available form and amount of the MMV Pathogen Box compounds. CLSI recommends using the disk diffusion test and agar dilution to determine MIC of antibiotics against N. gonorrhoeae (38). Although broth microdilution is a non-standard method for susceptibility testing, several studies have shown acceptable agreement between both methods (39 – 42). Therefore, we think it is reasonable to use broth microdilution for our initial purpose of compound screening, but confirmation of results using the disk diffusion test and agar dilution would be required for further study of these compounds.

Eight compounds, MMV676501 (Pbc 1), MMV002817 (Pbc 3), MMV688327 (Pbc 4, radezolid), MMV688508 (Pbc 5), MMV024937 (Pbc 6), MMV687798 (Pbc 7, levofloxacin), MMV021013 (Pbc 9), and MMV688978 (Pbc 10, auranofin), showed potent activity against two resistant strains of N. gonorrhoeae at MIC and MBC of ≤10 µM, with similar time-kill activity as ceftriaxone. Besides these eight compounds, MMV676388 (Pbc 2) and MMV272144 (Pbc 8) showed full activity against susceptible N. gonorrhoeae ATCC strains. The reference strains recorded lower MIC and MBC than the clinical strains. A molecular mechanism such as the reduced influx of drugs into the cell through transport proteins or increased efflux of drugs out of the cell via efflux pumps might account for the high MIC of N. gonorrhoeae active MMV Pathogen Box compounds in resistant clinical strains (32). All the eight compounds with activity in resistant N. gonorrhoeae strains were bactericidal and were either synergistic or additive with fractional inhibitory concentration index ranging between 0.40 and 1.8. Based on their MIC and time-kill kinetics, the four best compounds, MMV024937 (Pbc 6), MMV687798 (Pbc 7, levofloxacin), and MMV688978 (Pbc 10, auranofin), had an additive activity with ceftriaxone, suggesting their potential use as a combination therapy. A limitation of our study is that we only had access to a limited amount of compound which influenced the number of repeated measurements that we could perform. Assessment of MIC and MBC was done in triplicate while time-kill kinetics were assessed in duplicate measurement ( Tables S8 to S12). Variance in the repeated measurements was limited and within the acceptable range.

Our finding of auranofin activity against N. gonorrhoeae is consistent with the previous report of the activity of auranofin in MDR N. gonorrhoeae (35). After 4 hours, auranofin exhibited rapid killing and complete eradication of bacterial inoculum. There was a complete bactericidal activity and rapid eradication of a high bacterial inoculum after 6 hours with the combination of MMV688978 (Pbc 10, auranofin) and MMV024937 (Pbc 6). In a follow-up in vivo study by Elhassanny and colleagues (43), auranofin was found to significantly reduce N. gonorrhoeae from the vagina of infected mice (43). Further research of auranofin as a potential drug, alone or in combination, against N. gonorrhoeae is warranted.

Even though the two clinical strains were ciprofloxacin resistant, levofloxacin (MMV687798, Pbc 7) demonstrated bactericidal activity against N. gonorrhoeae strains. Levofloxacin is more active than ciprofloxacin against several organisms, including Streptococcus pneumoniae, Staphylococcus aureus, Bacteroides fragilis, and Clostridium species (44). Levofloxacin was highly active against several Gram-negative bacteria, including Neisseria meningitidis, Haemophilus influenzae, Proteus spp., Proteus mirabilis, Morganella morganii, and Enterobacter cloacae (45). Consistent with our study, N. gonorrhoeae was highly susceptible to levofloxacin at a concentration of ≤10 µM and showed additive effect with ceftriaxone and other NG active Pathogen Box compounds.

This study reported the activity of eight novel compounds from the Pathogen Box with potent activity against Neisseria gonorrhoeae. Six showed full activity in clinical strains. At 3× MIC, the drug molecules demonstrated bactericidal activity. MMV002817 (Pbc 3) and MMV024937 (Pbc 6) showed activity against Lymphatic filariasis-onchocerciasis and Plasmodium falciparum, respectively, in primary screen. The remaining compounds have shown activity against M. tuberculosis. The mechanism of action of MMV676501 (Pbc 1), MMV676388 (Pbc 2), MMV272144 (Pbc 8), and MMV021013 (Pbc 9) is unknown (27, 28, 46). It is possible that the compounds investigated in other organisms may result from differences in cellular targets that may be effective against resistant strains. Besides auranofin and levofloxacin, MMV024937 (Pbc 6) was one of the best compounds that rapidly eradicated a high N. gonorrhoeae bacterial inoculum (~1 × 10⁶) even at 1× MIC (Fig. 1; Fig. S4). After 6 hours, auranofin combined with MMV024937 (Pbc 6) completely eradicated the bacteria (Fig. 2).

The activity of the N. gonorrhoeae active Pathogen Box compounds is unknown in other Gram-negative bacteria. Few of the other Pathogen Box compounds have demonstrated activity against Gram-negative bacteria. MMV675968 (a diaminoquinazoline analog) inhibited different strains of A. baumannii, Escherichia coli, and Vibrio cholerae (30, 47, 48). Also, MMV687807 effectively inhibited the growth of V. cholerae (48).

Based on the data provided by MMV, the cytotoxicity profile of the N. gonorrhoeae active Pathogen Box compounds in human liver cells is promising (Table 1). Drug metabolism and pharmacokinetics (DMPK) of these compounds in mice have been studied following intravenous or orally by the Medicines for Malaria Venture group (https://www.mmv.org/mmv-open/pathogen-box/about-pathogen-box).

Since the pre-clinical pipeline remains largely empty of new agents that are likely to advance to development for gonorrhoea treatment (49), further studies on N. gonorrhoeae active compounds from MMV Pathogen Box are urgently needed. This study had a few limitations. First, as discussed, broth microdilution as non-standard method for susceptibility testing was used due to the form and amount of compound available in the MMV Pathogen Box. Testing of these compounds using the recommended CLSI methods is required in further studies. Second, an in vitro approach was used for initial testing instead of in silico or mechanistic approach to investigate the antimicrobial potential of 400 MMV Pathogen Box compounds against strains of N. gonorrhoeae (36). Also, the potential emergence of resistance of the active Pathogen Box compounds against N. gonorrhoeae in the time-kill assay was not explored (37).

Conclusions

In conclusion, we report 10 Pathogen Box compounds, including two reference drugs, auranofin and levofloxacin, that have promising in vitro antibacterial activity against N. gonorrhoeae strains. The activity of MMV Pathogen Box compounds has not been explored before against N. gonorrhoeae. This study provides additional resources for future anti-gonococcal drug research. Our study suggests that these compounds should be further investigated as potential novel options for primary or adjunctive therapy for the treatment of gonococcal infection.

MATERIALS AND METHODS

N. gonorrhoeae strains, antimicrobial susceptibility testing, and resistance mechanisms

N. gonorrhoeae ATCC 49226 and ATCC 19424 reference strains were purchased and used for quality control. Stored clinical strain 1 (SRS5471848) and clinical strain 2 (SRS5471840) were used; these had been collected from symptomatic male patients in Johannesburg, South Africa between March 2018 and April 2019 (13). Urethral swabs (Copan Diagnostics, Italy) were collected and immediately inoculated on New York City (NYC) agar medium and incubated at 35°C in a 5% CO₂ incubator for 24 hours. Presumptive colonies were identified using Gram staining, a rapid oxidase test, and the API NH system. This was followed by antibiotic susceptibility testing to azithromycin, ceftriaxone, cefixime, ciprofloxacin, penicillin G, and tetracycline using Etest. The MICs were interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. In the case of azithromycin, the epidemiological cut-off (ECOFF) value of 1.0 mg/L was used for interpretation (www.eucast.org). Genomic DNA was prepared using a High Pure PCR Template Preparation Kit (Roche, Germany). The pure DNA was further sequenced on the Illumina MiSeq (Illumina Inc., USA) platform to identify the various resistant genes or mechanisms as applied to the reference agent (13). Table 4 shows the antimicrobial profile and the mechanism of antibiotic resistance of N. gonorrhoeae clinical isolates.

Chemicals and preparation of chocolate agar and liquid broth for N. gonorrhoeae

GC base medium (BD Difco), BBL Haemoglobin Powder (Sigma-Aldrich), and BBL IsovitaleX Enrichment (BD Difco) were used to prepare chocolate agar. Brain heart infusion (Sigma-Aldrich), BBL IsovitaleX Enrichment (BD Difco), Agarose, and fetal bovine serum (Thermofisher Scientific) were used to prepare the fastidious broth. PrestoBlue High Sensitivity (HS) Cell Viability Reagent (Thermofisher Scientific) was used as a growth indicator (49, 50). Brain heart infusion broth (37.0 g/L) was supplemented with 5% fetal bovine serum, 1% IsovitaleX (gonococcal additive), and 1% agarose solution (0.75%) to prepare the liquid broth for N. gonorrhoeae.

Establishment of liquid broth and cultivation of N. gonorrhoeae

The liquid broth was prepared and distributed into 96-well flat-bottom microtiter plates, followed by inoculation with N. gonorrhoeae isolates. After incubation at 35°C under 5% CO₂ for 24 hours, N. gonorrhoeae grew well. PrestoBlue HS Cell Viability Reagent indicated macroscopic growth, and colony count ranged from 1.0 × 10⁴ to ~1.0 × 10⁷ and from 1.0 × 10⁶ to ~1.0 × 10¹¹ after 24 hours.

Preparation of Pathogen Box compounds

MMV provided the Pathogen Box in five (A–E) 96-well plates, with each plate consisting of 80 10 µL of 10 mM compounds. The plate mapping of the 400 compounds included in the Pathogen Box, including the biological data and DMPK data with chemical structures (as SMILES or with illustrations of the structures), can be found via the following link, covering the essential information on the compounds: https://www.mmv.org/mmv-open/pathogen-box/about-pathogen-box. The compounds were prepared/diluted according to the manufacturer’s instructions by dissolving the compounds in DMSO (Sigma Aldrich) and deionized distilled water to create a stock solution of 1 mM and stored at −80°C. The final concentration of DMSO (Sigma Aldrich) in all the assay wells was less than 1%. N. gonorrhoeae tolerance to 1% DMSO (Sigma Aldrich) was examined, and this concentration of solvent did not affect the bacteria growth/viability.

Determination of MIC and MBC of MMV Pathogen Box compounds by the broth dilution method

The MIC of MMV Pathogen Box compounds was determined using non-standard broth microdilution assay, as previously described (35, 38 – 42). All the 400 drug molecules, including reference drugs, were tested against clinical strain 1 (SRS5471848) and clinical strain 2 (SRS5471840) at 10 µM. After the primary evaluation, compounds that showed partial to full inhibition were selected and repeated in triplicate in a 96-well flat-bottom microtiter plate using a twofold broth microdilution assay at a concentration ranging from 0.156 to 10 µM to identify compounds with full activity and determine pathogen-specific MIC and MBC. These compounds were also tested against the susceptible N. gonorrhoeae ATCC 49226 strain. PrestoBlue HS Cell Viability Reagent was used as a growth indicator as previously described (50, 51). A change in color from blue to pink is an indication of bacterial growth. The lowest concentration with no color change was recorded as the MIC. Subsequently, an aliquot was taken from MIC assays where there is no visible growth and plated on chocolate agar. The MBC was determined as the lowest concentration that produces a 99.9% (3 log) decrease in visible bacterial growth. The experiment was repeated three times for only compounds which show activity in the primary evaluation.

Checkerboard-based determination of MMV Pathogen Box compounds synergies

The ability of the compounds to work in combination, and in conjunction with conventional antibiotic ceftriaxone, used in the treatment of gonorrhoea at a reduced MIC was assessed as previously described (52, 53). An overnight N. gonorrhoeae suspension equivalent to 1.0 McFarland standard was prepared and further diluted in a fresh broth until a bacterium inoculum of 5 × 10⁵ cfu/mL was achieved. Subsequently, the compounds and conventional drug ceftriaxone were added at different concentrations along with bacteria-containing media. Increasing concentrations of one compound were added to half the MIC of another compound to determine the lowest and best concentrations with synergy. The 96-well flat-bottom microtiter plates were incubated at 35°C for 24 hours in a 5% CO₂ incubator. The fractional inhibitory concentration index (FICI) was calculated for each compound used at the given concentration. The FICI was calculated using the formula, FICI= (MIC of agent A in combination)/(MIC of agent A alone) + (MIC of agent B in combination)/(MIC of agent B alone). A FICI value of ≤0.5 was considered as synergistic activity, between >0.5 and ≤1.25 as an additive activity, ≤4 as indifference, and >4.0 as antagonistic activity (54, 55).

Time-kill kinetics

To determine if the compounds that show activity against resistant N. gonorrhoeae strains are either bacteriostatic or bactericidal antibacterial in vitro, a time-kill assay was performed (56). Briefly, an overnight culture of N. gonorrhoeae was diluted in a fresh broth and incubated until the inoculum was ~5 × 10⁶ cfu/mL. Next, the bacterial solution was exposed to 3× MIC of the compounds. Azithromycin was used as a positive control, and DMSO served as a negative control. At 0, 2, 4, 6, 8, 10, 12, and 24 hours, an aliquot from each sample was serially diluted (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) and plated onto chocolate agar plates. Plates were incubated for 24 hours at 35°C in the presence of 5% CO₂ to determine the colony-forming unit count. If the initial bacterial cfu was reduced by at least 3 log₁₀ over 24 hours, the test compound was considered bactericidal, and <2 log reduction bacteriostatic.

ACKNOWLEDGMENTS

We acknowledge MMV for designing and supplying us with the Pathogen Box.

The student was financially supported by the University of Pretoria Doctoral Research Bursary.

We declare no conflicts of interest.

Contributor Information

Remco P. H. Peters, Email: remco.peters@up.ac.za.

Audrey Odom John, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA .

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aac.00348-23.

Figures S1 to S4. aac.00348-23-s0001.docx.

Supplementary figures

Click here for additional data file.^{(5.7MB, docx)}

DOI: 10.1128/aac.00348-23.SuF1

Tables S1 to S12. aac.00348-23-s0002.docx.

Supplementary tables

Click here for additional data file.^{(52.7KB, docx)}

DOI: 10.1128/aac.00348-23.SuF2

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figures S1 to S4. aac.00348-23-s0001.docx.

Supplementary figures

Click here for additional data file.^{(5.7MB, docx)}

DOI: 10.1128/aac.00348-23.SuF1

Tables S1 to S12. aac.00348-23-s0002.docx.

Supplementary tables

Click here for additional data file.^{(52.7KB, docx)}

DOI: 10.1128/aac.00348-23.SuF2

[B1] 1. Centers for Disease Control and Prevention . 2018. Sexually transmitted disease surveillance. Atlanta, GA. Available from: https://www.cdc.gov/nchhstp/newsroom/2019/2018-STD-surveillance-report.html [Google Scholar]

[B2] 2. World Health Organization . 2011. Emergence of multi-drug resistant Neisseria gonorrhoeae: threat of global rise in untreatable sexually transmitted infections. Available from: https://apps.who.int/iris/bitstream/handle/10665/70603/WHO_RHR_11.14_eng.pdf

[B3] 3. World Health Organization . 2012. Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae. World Health Organization Geneva, Switzerland. Available from: https://apps.who.int/iris/bitstream/handle/10665/44863/9789241503501_eng.pdf [Google Scholar]

[B4] 4. Walker CK, Sweet RL. 2012. Gonorrhea infection in women: prevalence, effects, screening, and management. Int J Womens Health 3:197. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Edwards JL, Apicella MA. 2004. The molecular mechanisms used by Neisseria gonorrhoeae to initiate infection differ between men and women. Clin Microbiol Rev 17:965–981, doi: 10.1128/CMR.17.4.965-981.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Tapsall JW. 2009. Neisseria gonorrhoeae and emerging resistance to extended-spectrum cephalosporins. Curr Opin Infect Dis 22:87–91. doi: 10.1097/QCO.0b013e328320a836 [DOI] [PubMed] [Google Scholar]

[B7] 7. Rice PA, Shafer WM, Ram S, Jerse AE. 2017. Neisseria gonorrhoeae: drug resistance, mouse models, and vaccine development. Annu Rev Microbiol 71:665–686. doi: 10.1146/annurev-micro-090816-093530 [DOI] [PubMed] [Google Scholar]

[B8] 8. Burnett AM, Anderson CP, Zwank MD. 2012. Laboratory-confirmed gonorrhea and/or chlamydia rates in clinically diagnosed pelvic inflammatory disease and cervicitis. Am J Emerg Med 30:1114–1117. doi: 10.1016/j.ajem.2011.07.014 [DOI] [PubMed] [Google Scholar]

[B9] 9. Little JW. 2006. Gonorrhea: update. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:137–143. doi: 10.1016/j.tripleo.2005.05.077 [DOI] [PubMed] [Google Scholar]

[B10] 10. WHO . 2011. Emergence of multi-drug resistant Neisseria gonorrhoeae: threat of global rise in untreatable sexually transmitted infections. Available from: https://apps.who.int/iris/bitstream/handle/10665/70603/WHO_RHR_11.14_eng.pdf

[B11] 11. Unemo M, Del Rio C, Shafer WM. 2016. Antimicrobial resistance expressed by Neisseria gonorrhoeae: a major global public health problem in the 21st century. Microbiol Spectr 4. doi: 10.1128/microbiolspec.EI10-0009-2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. 2012. High-level cefixime-and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 56:1273–1280. doi: 10.1128/AAC.05760-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Maduna LD, Kock MM, van der Veer BMJW, Radebe O, McIntyre J, van Alphen LB, Peters RPH. 2020. Antimicrobial resistance of Neisseria gonorrhoeae isolates from high-risk men in Johannesburg, South Africa. Antimicrob Agents Chemother 64:e00906-20. doi: 10.1128/AAC.00906-20 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Antimicrobial effects of Medicines for Malaria Venture Pathogen Box compounds on strains of Neisseria gonorrhoeae

Eric Mensah

P Bernard Fourie

Remco P H Peters

Roles

ABSTRACT

INTRODUCTION

RESULTS