Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp

To determine the antimicrobial activity of enacyloxin IIa and gladiolin against Neisseria gonorrhoeae and Ureaplasma spp.


Introduction
Antimicrobial resistance (AMR) is of growing concern among sexually transmitted pathogens. Neisseria gonorrhoeae diagnoses are increasing yearly which is ultimately putting a pressure on increased prescribing and maybe driving development of AMR (PHE 2019b). In many countries resistance to ciprofloxacin persists, high-level azithromycin resistance is present and resistance to thirdgeneration cephalosporins such as ceftriaxone has begun to emerge (Unemo et al. 2019). In 2018, the first description of extensively drug-resistant (XDR) N. gonorrhoeae was identified in the United Kingdom and subsequently in Australia, with all cases linked to recent travel to South East Asia Jennison et al. 2019). Further reports of XDR cases with ceftriaxone resistance and intermediate azithromycin resistance were reported in the UK from two women with recent travel to Ibiza, Spain, in which genomically identical strains belonging to the characterized FC428 were isolated (Eyre et al. 2019).
Ureaplasma spp. are unique genus of bacteria which have an essential requirement for urea in energy production and also have one of the smallest genomes of any free living organism (Glass et al. 2000). These bacteria are linked to non-gonococcal urethritis in men (Beeton et al. 2019) strongly associated with chorioamnionitis and subsequent preterm birth, (Sweeney et al. 2017) development of bronchopulmonary dysplasia, necrotizing enterocolitis and intraventricular haemorrhaging among preterm neonates (Viscardi 2014) and identified as a cause of infectious hyperammonemia among immunocompromised patients (Bharat et al. 2015). AMR is a significant challenge among these organisms due to substantial levels of intrinsic resistance owing to the absence of a cell wall and lack of metabolic pathways for de novo folic acid synthesis. Current treatment relies on macrolide, fluoroquinolone and tetracycline antibiotics, although acquired resistance is present to all antibiotics and prevalence varies depending on geographical location . The lack of a cell wall affords Ureaplasma spp. intrinsic resistance to all beta lactam and glycopeptide antibiotics, but also provides an excellent model for ruling out cellular targets of novel antimicrobials (Hillitt et al. 2017).
With the emergence of XDR N. gonorrhoeae and presence of macrolide resistance among ureaplasmas, which may complicate treatment of neonatal infections, there is a growing need to develop antibiotics with novel cellular targets to overcome current mechanisms of resistance. Enacyloxin IIa (Mahenthiralingam et al. 2011) and gladiolin (Song et al. 2017) are novel polyketide antibiotics isolated from Burkholderia bacteria. These antimicrobial compounds are of interest due to either their activity against novel bacterial targets not targeted by currently approved antibiotics, or ability to overcome current resistance as follows. Enacyloxin IIa inhibits elongation factor-Tu (EF-Tu) which has not yet been clinically exploited as an antibiotic target (Parmeggiani and Nissen 2006). The novel macrolide gladiolin inhibits the bacterial RNA polymerase, but is able to overcome resistance to other antibiotics such as rifampicin which has the same target (Song et al. 2017). The antibacterial capacity of these compounds have been examined against a number of bacteria including Mycobacterium tuberculosis, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii (Mahenthiralingam et al. 2011;Song et al. 2017), but to date have yet to be examined against either N. gonorrhoeae or any mollicute.
In this study, we examined the antimicrobial activity of enacyloxin IIa and gladiolin against a panel of susceptible and multidrug-resistant N. gonorrhoeae isolates from South Wales and characterized collections. Additionally, we sought to determine the activity of these compounds against the cell wall-free Ureaplasma spp. as a representative of the Mollicutes class.

Materials and methods
Neisseria gonorrhoeae and Ureaplasma spp. strain collection A total of 14 N. gonorrhoeae were examined in this study. Seven isolates (Gwent 1-7) were recent clinical isolates from South Wales, UK. The remaining seven comprised of characterized susceptible and multidrug-resistant isolates NCTC 8375, NCTC 13798, NCTC 13799, NCTC 13477 (WHO F), NCTC 13818 (WHO V), NCTC 13820 (WHO X) and NCTC 13822 (WHO Z) (Unemo et al. 2016). Neisseria gonorrhoeae were propagated on Brain Heart Infusion (BHI) agar (Sigma, Dorset, UK) supplemented with 5% lysed horse blood (TCS Biosciences, Buckingham, UK). All cultures were incubated at 37°C in the presence of CO 2 .

Preparation of enacyloxin IIa and gladiolin
Enacyloxin IIa and gladiolin were prepared from Burkholderia gladioli strains BCC1701 and BCC0238 respectively. Both antibiotics were induced by growing Burkholderia on solidified basal salts medium with 4 g l À1 glycerol (BSM-G) for 72 h at 30°C and extracted using dichloromethane (Mullins et al. 2019). Extracts were concentrated using a Buchi Rotavapor R-3 system, eluted in 60% (v/v) acetonitrile and purified by preparative HPLC (Waters Autopurification HPLC system fitted with a XSelect CSH C18, 5 µm OBD, 199 1009 mm column). Fractions were collected at 360 and 230 nm for enacyloxin IIa and gladiolin respectively pooled and dried to a powder by a combination of vacuum centrifugation and freeze drying. Powdered aliquots of each antibiotic were stored at À80°C prior to dissolving in dimethyl sulphoxide to create working stocks for antimicrobial susceptibility testing.

Antimicrobial susceptibility testing of Ureaplasma spp
The susceptibility of Ureaplasma spp. to antimicrobials was determined by broth microdilution following the Clinical and Laboratory Standards Institute approved guidelines for testing human mycoplasmas (CLSI 2011). In brief, doubling dilutions of the test antimicrobial were prepared in USM within microtitre plates. Ureaplasma were added to each well giving a final concentration of 10 4 -10 5 colour changing units per ml. Plates were sealed and subsequently incubated at 37°C under ambient air. MIC values were determined as the lowest concentration of antimicrobial to inhibit colour change at the point at which the positive control well gave a colour change. Growth curve assays were set up in an identical way with the modification of static incubation within a Tecan Infinite M200 spectrophotometer. Plates were sealed and incubated at 37°C for 20 h with readings taken at 550 nm were taken every 20 min.

Antimicrobial susceptibility testing of N. gonorrhoeae
Agar dilution was used to determine the susceptibility of N. gonorrhoeae to the test antimicrobials. Briefly, doubling dilutions of antimicrobials were prepared in agar plates containing BHI supplemented with 5% defibrinized horse blood giving a final concentration range from 32 to 0Á015 mg l À1 . Neisseria gonorrhoeae colonies were suspended in saline to a 0Á5 McFarland Standard. A multipoint inoculator was used to inoculate plates with 1 µl of bacterial suspension in duplicate. Plates were incubated for 30 h at 37°C in the presence of CO 2 . For quality assurance purposes, MIC values for the WHO strains were confirmed using the method described.

Activity of enacyloxin IIa and gladiolin against N. gonorrhoeae
The activity of enacyloxin IIa and gladiolin against N. gonorrhoeae was determined using an agar dilution assay. The MIC range for enacyloxin IIa against N. gonorrhoeae was lower than that of gladiolin (0Á015-0Á06 mg l À1 versus 1-2 mg l À1 respectively) ( Table 1). The presence of defined high-level azithromycin resistance (WHO V) or ceftriaxone resistance (WHO X) in a background of ciprofloxacin and tetracycline resistance had no impact on the MIC compared with fully susceptible strains.

Activity of enacyloxin IIa and gladiolin against Ureaplasma spp
Using broth microdilution the MIC range of enacyloxin IIa against Ureaplasma spp. was determined to be  between 4 and 32 mg l À1 whereas the MIC for gladiolin was greater than 32 mg l À1 ( Table 2). The effect of enacyloxin IIa on the growth kinetics of the macrolide resistant U. parvum strain UHWO10 was determined by spectrophotometry (Fig. 1). There was a clear dose-dependent inhibition of growth over time with enacyloxin IIa, which was not seen with gladiolin or erythromycin.

Discussion
Antibiotic resistance among sexually transmitted pathogens is of growing concern. In 2018, the first reports of XDR N. gonorrhoeae with ceftriaxone and high-level azithromycin resistance were noted in England and shortly after in Australia (Jennison et al. 2019). Although numbers of XDR cases are limited, recent data from the  Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) identified a drift in all antimicrobials tested away from susceptible ranges (PHE 2019a). In light of these data there is a need to invest and develop novel antimicrobial compounds. Enacyloxin represents a novel class of antibiotic for future development (Mahenthiralingam et al. 2011). The MIC values for enacyloxin IIa against N. gonorrhoeae were lower than for front line agents azithromycin and ceftriaxone. The MIC values were substantially lower than those previously reported for A. baumannii (3 mg l À1 ) and Pseudomonas aeruginosa (100 mg l À1 ; Mahenthiralingam et al. 2011). Of particular interest was the activity of this compound against the multidrug-resistant strains with documented resistance to azithromycin, ceftriaxone, tetracycline and ciprofloxacin. By inhibiting protein synthesis via interactions with EF-TU, enacyloxin can bypass current mechanisms of resistance. In addition, the recent finding that the chain release mechanism required enacyloxin biosynthesis can be manipulated to produce novel analogues, opens up multiple possibilities for the clinical development of this antibiotic (Masschelein et al. 2019). Although the activity of gladiolin was not as pronounced as enacyloxin IIa, it was again comparable to that of azithromycin and ceftriaxone. Ureaplasma spp. are recognized pathogens associated with in utero infection, infection among immunocompromised patients and mounting evidence as pathogens in sexual health (Bharat et al. 2015;Sweeney et al. 2017;Beeton et al. 2019). Intrinsic resistance to many antimicrobials and emergence of acquired resistance makes treatment complicated, especially in the context of neonatal infection in which tetracyclines and fluoroquinolones are contraindications. Endpoint MIC readings showed that MIC values ranged from 4 to 32 mg l À1 for enacyloxin IIa and >32 for gladiolin. These values were comparable to those previously seen for A. baumannii (Mahenthiralingam et al. 2011). Due to the lack of quantifiable turbidity when growing ureaplasma in broth culture, a colorimetric and kinetic based method was used to observe any dose-dependent impact of antimicrobials. For enacyloxin IIa there was a clear dose dependent inhibition of growth which was absent in the presence of gladiolin as well erythromycin to which the UHWO10 is resistant to. Enaycyloxin is known to bind serum proteins and therefore the presence of 20% serum in the ureaplasma media may have chelated out active drug explaining the higher MIC values. Reasons for the lack of gladiolin activity is unknown, but may be linked to the lack of antimicrobial activity against ureaplasmas seen for other RNA polymerase inhibiting antibiotics such as rifampin (Waites et al. 2005).
In conclusion, this study demonstrates the potential for enacyloxin IIa and gladiolin as future therapeutics against N. gonorrhoeae, with particular reference to multidrug-resistant strains. These data suggest further investigation and development of this compound as a potential for treating XDR N. gonorrhoeae and mycoplasmas.