Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES - December, 2018

Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES

December, 2018



Reviews and Commentary

Neisseria gonorrhoeae - Rising Infection Rates, Dwindling Treatment Options.

Blank S, Daskalakis DC. (Full Text)

N Engl J Med. 2018 Nov 8;379(19):1795-1797. doi: 10.1056/NEJMp1812269.



Antimicrobial Resistance in Neisseria gonorrhoeae: Proceedings of the STAR Sexually Transmitted Infection-Clinical Trial Group Programmatic Meeting.

Cristillo AD, Bristow CC, Torrone E, Dillon JA, Kirkcaldy RD, Dong H, Grad YH, Nicholas RA, Rice PA, Lawrence K, Oldach D, Shafer WM, Zhou P, Wi TE, Morris SR, Klausner JD. (Full Text)

Sex Transm Dis. 2018 Oct 22. doi: 10.1097/OLQ.0000000000000929. [Epub ahead of print]

Abstract

The goal of the Sexually Transmitted Infection Clinical Trial Group's (STI-CTG) Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, non-profit and industry to discuss the current state of research, gaps and challenges in research and technology as well as priorities and new directions to address the continued emergence of multi-drug resistant NG infections. Topics discussed at the meeting, that will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing (WGS) and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG. (ii) The US National Strategy for combating antimicrobial resistant bacteria. (iii) Surveillance needs, challenges and novel technologies. (iv) Plasmid- and chromosomally-mediated mechanisms of AMR in NG, (v) Novel therapeutic (e.g., sialic acid analogs, FH/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (e.g., peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives and collaborations across academic and commercial research and public health programs.

Epidemiology and Surveillance 

Multidrug-resistant Neisseria gonorrhoeae isolate, belonging to the internationally spreading Japanese FC428 clone, with ceftriaxone resistance and intermediate resistance to azithromycin, Ireland, August 2018.

Golparian D, Rose L, Lynam A, Mohamed A, Bercot B, Ohnishi M, Crowley B, Unemo M. (Full Text)

Euro Surveill. 2018 Nov;23(47). doi: 10.2807/1560-7917.ES.2018.23.47.1800617.

Abstract

We describe a multidrug-resistant Neisseria gonorrhoeae urethritis case with ceftriaxone resistance and azithromycin intermediate resistance in a heterosexual man in Ireland, August 2018. Whole-genome sequencing showed that the isolate IR72 belongs to the internationally spreading multidrug-resistant ceftriaxone-resistant FC428 clade, initially described in Japan in 2015. IR72 was assigned MSLT ST1903, NG-MAST ST17842 and NG-STAR type 1133, including the ceftriaxone resistance-mediating penA-60.001. Global awareness of spreading ceftriaxone-resistant gonococcal strains that threaten recommended dual therapies is essential.



Establishment of a gonococcal antimicrobial surveillance programme (GASP), in accordance with WHO standards, in Côte d'Ivoire, Western Africa, 2014-2017.

Yéo A, Kouamé-Blavo B, Kouamé CE, Ouattara A, Yao AC, Gbedé BD, Bazan F, Faye-Ketté H, Dosso M, Wi T, Unemo M(Full Text)

Sex Transm Dis. 2018 Nov 19. doi: 10.1097/OLQ.0000000000000943. [Epub ahead of print]

Abstract

BACKGROUND:

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is compromising the treatment of gonorrhea globally. Recent AMR data are extremely limited in Africa, and mainly totally lacking in Western Africa including Côte d'Ivoire. This study i) established a quality-assured gonococcal antimicrobial surveillance programme (GASP), according to WHO quality criteria, ii) investigated the AMR to eight therapeutic antimicrobials in gonococcal isolates from 2014 to 2017, and iii) provided evidence for updating the national STD syndromic management guidelines in Côte d'Ivoire.

METHODS:

During 2014-2017, gonococcal isolates were obtained from sexually active symptomatic or asymptomatic males and females in 14 sites in Côte d'Ivoire. It was a special focus on symptomatic males, and their sexual partners, due to the higher culture positivity rates in symptomatic males. Patient metadata were collected, including age, gender, sexual orientation and symptoms. Minimum inhibitory concentrations (MICs) of eight antimicrobials were determined by Etest and interpreted using EUCAST breakpoints. β-lactamase production was detected using cefinase disks.

RESULTS:

The level of resistance, examining 212 gonococcal isolates, was as follows: 84.9% to tetracycline, 68.9% to benzylpenicillin, 62.7% to ciprofloxacin, 6.1% to azithromycin, and 1.4% to gentamicin. All isolates were susceptible to ceftriaxone, cefixime and spectinomycin.

CONCLUSION:

We provide the first gonococcal AMR data, quality assured according to WHO standards, from Côte d'Ivoire since more than 20 years. The high ciprofloxacin resistance, which informed a revision of the national syndromic management guideline during study, and relatively high resistance to azithromycin demand an improved GASP and increased awareness when prescribing treatment in Côte d'Ivoire.



Association between intensity of STI screening and development of antimicrobial resistance in N. gonorrhoeae in 12 cities in the USA: An ecological study.

Kenyon CR. (Full Text)

Version 2. F1000Res. 2018 Aug 10 [revised 2018 Sep 27];7:1237. doi: 10.12688/f1000research.15569.2. eCollection 2018.

Abstract

In this study, we assessed if there was a city-level association between sexually transmitted infection (STI) screening intensity in men who have sex with men and antimicrobial sensitivity in Neisseria gonorrhoeae in the United States, 2007 to 2013.  We found positive associations between STI screening intensity and increases in minimum inhibitory concentrations for cefixime and azithromycin, but not ceftriaxone.



Antimicrobial resistance and molecular characterization of Neisseria gonorrhoeae isolates in Fukuoka, Japan, from 1996 to 2016.

Tanaka M, Furuya R, Kobayashi I, Kanesaka I, Ohno A, Katsuse AK. (Full Text)

J Glob Antimicrob Resist. 2018 Nov 15. pii: S2213-7165(18)30227-3. doi: 10.1016/j.jgar.2018.11.011. [Epub ahead of print]



Abstract

OBJECTIVES:

We examined the antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates obtained from 1996 -2005 (n=200) and 2008-2016 (n=200) in Fukuoka, Japan.

METHODS:

MICs were determined by agar dilution and sequence types (STs) were examined using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Sequencing of the major extended-spectrum cephalosporin (ESC) resistance determinants (penA, mtrR, and ponA) was performed.

RESULTS:

Increases in the proportion of gonococci with decreased susceptibility or resistance to cefixime (from 18% in 1996-2005 to 46% in 2008-2016) and ceftriaxone (from 2.5% to 4%) were observed. Gonococcal isolates also showed increases in resistance to ciprofloxacin and azithromycin. The 4 most prevalent NG-MAST STs with a multi-drug resistance phenotype were ST2958 (n=18), ST1407 (n=14), ST 6798 (n=12), and ST4015 (n=10). The number of isolates belonging to the 4 STs rose from the first to the second period. Among the 54 isolates belonging to the 4 major STs, 42 (77.8%) contained a penA mosaic allele and 12 (22.2%) a penA non-mosaic allele. The sequence pattern types in the 42 isolates with a penA mosaic allele included type X (64.3%), type XXXIV (33.3%), and a novel pattern type (2.4%). In contrast, all 12 isolates with the penA non-mosaic allele included the sequence pattern type V (100%).

CONCLUSION:

N. gonorrhoeae isolates with decreased susceptibilities or resistance to ESC have increased over the years, in Fukuoka, Japan. Four major STs with a multi-drug resistance phenotype were identified. These isolates contained a penA mosaic allele or a non-mosaic allele in PBP2.



Antimicrobial resistance and molecular characteristics of Neisseria gonorrhoeae isolates from MSM.

Calado J, Castro R, Lopes Â, Campos MJ, Rocha M, Pereira F. (Full Text)

Int J Infect Dis. 2018 Nov 6. pii: S1201-9712(18)34578-8. doi: 10.1016/j.ijid.2018.10.030. [Epub ahead of print]

Abstract

OBJECTIVES:

To analyze the susceptibility of Neisseria gonorrhoeae isolates to penicillin (Pen), cefixime (Cfm), ceftriaxone (Cro), tetracycline (Tet), ciprofloxacin (Cip), azithromycin (Azm) and spectinomycin (Spt). To verify the presence of mutations in resistant genes.

METHODS:

Antibiotic susceptibility testing was carried out by Etest method in 30N. gonorrhoeae isolates collected from MSM population. PCR and DNA sequencing were performed to identify mutations within penA, mtrR, gyrA and parC genes in intermediate or full resistance isolates.

RESULTS:

N. gonorrhoeae isolates showed intermediate or full resistance to Pen (73%), Cfm (3%), Tet (60%), Cip (37%) and Azm (13%). One isolate CfmR presented a PBP2 mosaic XXXIV. All PenI and PenR isolates (except at PBP2 mosaic) presented a D345a in PBP2 and all CipR isolates had a S91F at gyrA gene together with mutations at parC gene. All intermediate or full resistance isolates to substrates of MtrCDE efflux pump had an A39T or G45D mutation in mtrR gene or an adenine deletion within the mtrR promoter. One isolate presented a N. meningitidis-like mtrR promoter sequence.

CONCLUSIONS:

The results of this study are consisting with the findings of other authors and reinforce the importance of developing new therapeutic options in a short time period.



Whole-Genome Sequencing of Russian Neisseria gonorrhoeae Isolates Related to ST 1407 Genogroup.

Kubanov AA, Runina AV, Chestkov AV, Kudryavtseva AV, Pekov YA, Korvigo IO, Deryabin DG. (Full Text)

Acta Naturae. 2018 Jul-Sep;10(3):68-76.

Abstract

The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for β-lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.



Complete ciprofloxacin resistance in gonococcal isolates in an urban Ugandan clinic: findings from a cross-sectional study.

Mabonga E, Parkes-Ratanshi R, Riedel S, Nabweyambo S, Mbabazi O, Taylor C, Gaydos C, Manabe YC. (Full Text)

Int J STD AIDS. 2018 Nov 4:956462418799017. doi: 10.1177/0956462418799017. [Epub ahead of print]

Abstract

Antimicrobial resistance (AMR) to gonorrhoea is a threat to global health security. There have been concerns expressed that countries with high rates of disease have poor surveillance. The objectives of the study were to determine the AMR patterns of Neisseria gonorrhoeae clinical isolates to antimicrobial agents in patients with HIV or high risk of HIV acquisition, to compare the concordance of disk diffusion and agar dilution as methods for determining AMR to N. gonorrhoeae, and to describe methodological challenges to carrying out AMR testing. The study was conducted at an HIV outpatient service for at-risk populations and an outreach clinic for commercial sex workers in Kampala. Patients were offered a sexually transmitted infection screen using a polymerase chain reaction (PCR)-based assay. Samples positive for gonorrhoea were cultured. Antimicrobial susceptibility testing was performed using disk diffusion and isolates were sent to a reference laboratory for agar dilution direct susceptibility testing. Five hundred and seventy-five patients were screened. There were 33 (5.7%) patients with gonorrhoea detected by PCR. Of the 16 viable N. gonorrhoeae isolates, 100% were resistant to ciprofloxacin and tetracycline by disk diffusion and 31% exhibited reduced susceptibility to ceftriaxone and cefixime. By agar dilution, 100% of isolates were resistant to ciprofloxacin and all isolates were susceptible to ceftriaxone and cefixime. There was concordance between disk diffusion and agar dilution for ciprofloxacin and tetracycline resistance and a significant discordance for third-generation cephalosporins. More than half the women with gonorrhoea were asymptomatic and represent a potential reservoir for ongoing transmission. AMR testing of N. gonorrhoeae isolates is needed to ensure optimal treatment and prevention of antibiotic resistance progression.



The first year of the global Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) in Bangkok, Thailand, 2015-2016.

Sirivongrangson P, Girdthep N, Sukwicha W, Buasakul P, Tongtoyai J, Weston E, Papp J, Wi T, Cherdtrakulkiat T, Dunne EF; EGASP Thailand Workgroup. (Full Text)

PLoS One. 2018 Nov 9;13(11):e0206419. doi: 10.1371/journal.pone.0206419. eCollection 2018.

Abstract

Antimicrobial-resistant Neisseria gonorrhoeae (NG) infection is a global public health threat, and there is a critical need to monitor patterns of resistance and risk factors. In collaboration with the World Health Organization (WHO), the U.S. Centers for Disease Control and Prevention (CDC), and the Thailand Department of Disease Control (DDC), Ministry of Public Health (MoPH) implemented the first Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP) in November 2015. Men presenting with urethritis at two clinical settings in Bangkok, Thailand (Bangrak Hospital [BH] and Silom Community Clinic @TropMed [SCC @TropMed]) provided demographic and behavioral information and had a urethral swab for Gram's stain and NG culture collected. The NG isolates were evaluated for antimicrobial susceptibility by the Epsilometer test (Etest) to determine minimum inhibitory concentrations (MICs) for cefixime (CFM), ceftriaxone (CRO), azithromycin (AZI), gentamicin (GEN), and ciprofloxacin (CIP). From November 2015 -October 2016, 1,102 specimens were collected from 1,026 symptomatic men; 861 (78.1%) specimens were from BH and 241 (21.9%) specimens were from SCC @TropMed. Among the 1,102 specimens, 582 (52.8%) had intracellular Gram-negative diplococci and 591 (53.6%) had NG growth (i.e., NG infection); antimicrobial susceptibility testing (AST) was performed on 590 (99.8%) NG isolates. Among all symptomatic men, 293 (28.6%) had sex with men only, 430 (41.9%) were ages 18-29 years, 349 (34.0%) had antibiotic use in the last 2 weeks, and 564 (55.0%) had NG infection. Among 23 men with repeat NG infection during this first year of surveillance, 20 (87.0%) were infected twice, 2 (8.7%) were infected three times, and 1 (4.3%) was infected more than four times. All NG isolates were susceptible to CFM and CRO, and had MICs below 2 μg/mL for AZI and below 16 μg/mL for GEN. Overall, 545 (92.4%) isolates were resistant to CIP. This surveillance activity assessed individual patients, and included demographic and behavioral data linked to laboratory data. The inclusion of both individual and laboratory information in EGASP could help identify possible persistent infection and NG treatment failures. Expansion of EGASP to additional global settings is critical to assess trends and risk factors for NG, and to monitor for the emergence of resistance.



Increasing prevalence of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone and resistance to azithromycin in Hangzhou, China (2015-17).

Yan J, Xue J, Chen Y, Chen S, Wang Q, Zhang C, Wu S, Lv H, Yu Y, van der Veen S. (Full Text)

J Antimicrob Chemother. 2018 Oct 16. doi: 10.1093/jac/dky412. [Epub ahead of print]

Abstract

OBJECTIVES:

Development of resistance in Neisseria gonorrhoeae to ceftriaxone monotherapy or ceftriaxone plus azithromycin dual therapy is a global public health concern. The aim of this study was to analyse the trend in antimicrobial resistance in Hangzhou, China, over the period 2015-17.

METHODS:

In total, 379 clinical isolates were collected from seven hospitals and antimicrobial susceptibility was determined using the agar dilution method. Isolates showing resistance to ceftriaxone, azithromycin or cefixime were analysed for the presence of resistance determinants. STs were determined with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method and phylogenetic analysis and strain clustering was determined using porB and tbpB sequences.

RESULTS:

Ceftriaxone resistance, decreased susceptibility to ceftriaxone and azithromycin resistance were observed in 3%, 17% and 21% of the isolates, respectively. This resulted in 5% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Importantly, resistance levels to ceftriaxone and azithromycin increased over the study period, resulting in 5% ceftriaxone resistance, 27% decreased susceptibility to ceftriaxone and 35% azithromycin resistance in 2017 and 11% of the isolates showing both decreased susceptibility to ceftriaxone and azithromycin resistance. Phylogenetic and cluster analysis showed the emergence and expansion in 2017 of a clonally related cluster containing strains with high abundance of decreased susceptibility to ceftriaxone and/or cefixime, which was related to the presence of the mosaic penA allele X. Co-resistance to azithromycin was also observed in this cluster.

CONCLUSIONS:

Our findings have major implications for the future reliability of ceftriaxone monotherapy and ceftriaxone plus azithromycin dual therapy in China.

Novel detection strategies and diagnostics

 

Multiplex TaqMan real-time PCR platform for detection of Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone.

Zhao L, Liu A, Li R, Zhao S. (Full Text)

Diagn Microbiol Infect Dis. 2018 Oct 31. pii: S0732-8893(18)30555-8. doi: 10.1016/j.diagmicrobio.2018.10.013.  [Epub ahead of print]

Abstract

A multiplex TaqMan real-time PCR platform was developed in this study for combined detection of opa and/or porA genes (identification of N. gonorrhoeae) and the key mutations (Ala501Val/Thr/Pro, and/or Gly545Ser) in penicillin-binding protein 2 (PBP2) associated with decreased susceptibility to extended-spectrum cephalosporins (ESCs). Firstly, the specificities of the TaqMan probes/primers for the multiplex TaqMan real time PCR platform were confirmed by Basic Local Alignment Search Tool (BLAST) analysis. Then the multiplex PCR platform was performed on 77 isolates with decreased susceptibility to ceftriaxone (CRO) and 100 isolates with full susceptibility to CRO under universal optimized reaction conditions. As a result, based on cultivation-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility testing in vitro, the multiplex platform had a sensitivity of 100% and a specificity of 95.0% for identifying cultured isolates of Neisseria gonorrhoeae (N. gonorrhoeae, NG, GC) with decreased susceptibility to CRO. When directly screening N. gonorrhoeae with decreased susceptibility to CRO from clinical urogenital swabs, the multiplex platform offered a sensitivity of 96.1% and a specificity of 95.0%. Therefore, on the basis of sample culture and antimicrobial susceptibility testing in vitro, the multiplex TaqMan real time PCR platform has been proven to be a sensitivity of 100% and a specificity of 95.0% useful tool for screening cultured isolates of N. gonorrhoeae with decreased susceptibility to CRO, which can be finished within 2 days.


 

Identification and expression analysis of ceftriaxone resistance-related genes in Neisseria gonorrhoeae integrating RNA-Seq data and qRT-PCR validation.

Zhao YH, Qin XL, Yang JY, Liao YW, Wu XZ, Zheng HP. (Full Text)

J Glob Antimicrob Resist. 2018 Oct 12. pii: S2213-7165(18)30197-8. doi: 10.1016/j.jgar.2018.10.008. [Epub ahead of print]

Abstract

OBJECTIVES:

The aim of the present study was to identify the ceftriaxone resistance-related genes of N. gonorrhoeae.

METHODS:

We compared the differences in gene expression between the susceptibility (MIC=0.002-0.004μg/ml) and decreased susceptibility (MIC=0.125-0.5μg/ml) of isolates to ceftriaxone using RNA sequencing (RNA-Seq).

RESULTS:

The total RNAs of 10 clinical isolates were used to make libraries and generated an average of 24.07 Mb reads per sample; these were assembled into 1871 mRNA genes. Moreover, 21 differentially expressed genes (DEGs) were found between the two groups with fold change of >2 (P <0.05), wherein 11 were upregulated and 10 were downregulated. Furthermore, all DEGs were verified by qRT-PCR, which detected 25 clinical isolates with decreased susceptibility and 21 susceptible strains against ceftriaxone. Additionally, 7 DEGs revealed the relative expression levels by 2-DΔCt and showed a statistical significance (P≤0.05). Analysis of GO terms and KEGG pathway for functional enrichment showed that 6 DEGs were related to the cellular component and 1 DEG was related to the biosynthesis of antibiotics, and these results might be related with ceftriaxone resistance.

CONCLUSIONS:

searching the ceftriaxone resistance-related genes of N. gonorrhoeae is necessary for the high morbidity and antimicrobial resistance (AMR) of N. gonorrhoeae, especially in its eventual resistance to third-generation extended-spectrum cephalosporins (ESCs), namely, cefixime and ceftriaxone. Moreover, this reported provides a new direction for the study and control of ceftriaxone-resistant in N. gonorrhoeae.

Novel antibiotics, mechanisms, vaccine, and other advances

 

Single-Dose Zoliflodacin (ETX0914) for Treatment of Urogenital Gonorrhea.

Taylor SN, Marrazzo J, Batteiger BE, Hook EW 3rd, Seña AC, Long J, Wierzbicki MR, Kwak H, Johnson SM, Lawrence K, Mueller J. (Full Text)

N Engl J Med. 2018 Nov 8;379(19):1835-1845. doi: 10.1056/NEJMoa1706988.

Abstract

BACKGROUND:

Antibiotic-resistant Neisseria gonorrhoeae has prompted the development of new therapies. Zoliflodacin is a new antibiotic that inhibits DNA biosynthesis. In this multicenter, phase 2 trial, zoliflodacin was evaluated for the treatment of uncomplicated gonorrhea.

METHODS:

We randomly assigned eligible men and women who had signs or symptoms of uncomplicated urogenital gonorrhea or untreated urogenital gonorrhea or who had had sexual contact in the preceding 14 days with a person who had gonorrhea to receive a single oral dose of zoliflodacin (2 g or 3 g) or a single 500-mg intramuscular dose of ceftriaxone in a ratio of approximately 70:70:40. A test of cure occurred within 6±2 days after treatment, followed by a safety visit 31±2 days after treatment. The primary efficacy outcome measure was the proportion of urogenital microbiologic cure in the microbiologic intention-to-treat (micro-ITT) population.

RESULTS:

From November 2014 through December 2015, a total of 179 participants (167 men and 12 women) were enrolled. Among the 141 participants in the micro-ITT population who could be evaluated, microbiologic cure at urogenital sites was documented in 55 of 57 (96%) who received 2 g of zoliflodacin, 54 of 56 (96%) who received 3 g of zoliflodacin, and 28 of 28 (100%) who received ceftriaxone. All rectal infections were cured in all 5 participants who received 2 g of zoliflodacin and all 7 who received 3 g, and in all 3 participants in the group that received ceftriaxone. Pharyngeal infections were cured in 4 of 8 participants (50%), 9 of 11 participants (82%), and 4 of 4 participants (100%) in the groups that received 2 g of zoliflodacin, 3 g of zoliflodacin, and ceftriaxone, respectively. A total of 84 adverse events were reported: 24 in the group that received 2 g of zoliflodacin, 37 in the group that received 3 g of zoliflodacin, and 23 in the group that received ceftriaxone. According to investigators, a total of 21 adverse events were thought to be related to zoliflodacin, and most such events were gastrointestinal.

CONCLUSIONS:

The majority of uncomplicated urogenital and rectal gonococcal infections were successfully treated with oral zoliflodacin, but this agent was less efficacious in the treatment of pharyngeal infections. (Funded by the National Institutes of Health and Entasis Therapeutics; ClinicalTrials.gov number, NCT02257918 .).

 

Antimicrobial peptide LL-37 and its truncated forms, GI-20 and GF-17, exert spermicidal effects and microbicidal activity against Neisseria gonorrhoeae.

Kiattiburut W, Zhi R, Lee SG, Foo AC, Hickling DR, Keillor JW, Goto NK, Li W, Conlan W, Angel JB, Wang G, Tanphaichitr N. (Full Text)

Hum Reprod. 2018 Dec 1;33(12):2175-2183. doi: 10.1093/humrep/dey315.

Abstract

STUDY QUESTION:

Do the truncated LL-37 peptides, GI-20 and GF-17, have spermicidal activity and microbicidal effects on the sexually transmitted infection (STI) pathogen Neisseria gonorrhoeae with equivalent potency to LL-37?

SUMMARY ANSWER:

GI-20 and GF-17 exhibited spermicidal effects on both mouse and human sperm as well as microbicidal action on N. gonorrhoeae with the same efficacy as LL-37.

WHAT IS KNOWN ALREADY:

The antimicrobial peptide LL-37 exerts microbicidal activity against various STI pathogens as well as spermicidal effects on both mouse and human sperm.

STUDY DESIGN, SIZE, DURATION:

Spermicidal activities of GI-20 and GF-17 were evaluated in vitro in mouse and human sperm and in vivo in mice. Finally, in vitro antimicrobial effects of LL-37, GI-20 and GF-17 on an STI pathogen, N. gonorrhoeae were determined. All experiments were repeated three times or more. In particular, sperm samples from different males were used on each experimental day.

PARTICIPANTS/MATERIALS, SETTING, METHODS:

The plasma membrane integrity of peptide-treated sperm was assessed by cellular exclusion of Sytox Green, a membrane impermeable fluorescent DNA dye. Successful mouse in vitro fertilization was revealed by the presence of two pronuclei in oocytes following co-incubation with capacitated untreated/peptide-pretreated sperm. Sperm plus each peptide were transcervically injected into female mice and the success of in vivo fertilization was scored by the formation of 2-4 cell embryos 42 h afterward. Reproductive tract tissues of peptide pre-exposed females were then assessed histologically for any damage. Minimal inhibitory/bactericidal concentrations of LL-37, GI-20 and GF-17 on N. gonorrhoeae were determined by a standard method.

MAIN RESULTS AND THE ROLE OF CHANCE:

Like LL-37, treatment of sperm with GI-20 and GF-17 resulted in dose-dependent increases in sperm plasma membrane permeabilization, reaching the maximum at 18 and 3.6 μM for human and mouse sperm, respectively (P < 0.0001, as compared with untreated sperm). Mouse sperm treated with 3.6 μM GI-20 or GF-17 did not fertilize oocytes either in vitro or in vivo. Moreover, reproductive tract tissues of female mice pre-exposed to 3.6 μM GI-20 or GF-17 remained intact with no lesions, erosions or ulcerations. At 1.8-7.2 μM, LL-37, GI-20 and GF-17 exerted bactericidal effects on N. gonorrhoeae.

LARGE SCALE DATA:

N/A.

LIMITATIONS, REASONS FOR CAUTION:

Direct demonstration of the inhibitory effects of GI-20 and GF-17 on human in vitro and in vivo fertilization cannot be performed due to ethical issues.

WIDER IMPLICATIONS OF THE FINDINGS:

Like LL-37, GI-20 and GF-17 acted as spermicides and microbicides against N. gonorrhoeae, without adverse effects on female reproductive tissues. With lower synthesis costs, GI-20 and GF-17 are attractive peptides for further development into vaginal spermicides/microbicides.

STUDY FUNDING/COMPETING INTEREST(S):

This work was supported by Canadian Institutes of Health Research (MOP119438 and CCI82413 to N.T.) and NIH (R01 AI105147 to G.W.). There are no competing interests to declare.



Neisseria gonorrhoeae Exposed to Sublethal Levels of Hydrogen Peroxide Mounts a Complex Transcriptional Response.

Quillin SJ, Hockenberry AJ, Jewett MC, Seifert HS.  (Full Text)

mSystems. 2018 Oct 2;3(5). pii: e00156-18. doi: 10.1128/mSystems.00156-18. eCollection 2018 Sep-Oct.

Abstract

Neisseria gonorrhoeae mounts a substantial transcriptional program in response to hydrogen peroxide (HP), a prominent reactive oxygen species (ROS) encountered during infection. We tested which strain FA1090 genes show differential transcript abundance in response to sublethal amounts of HP to differentiate HP-responsive signaling from widespread cellular death and dysregulation. RNA sequencing (RNA-Seq) revealed that 150 genes were significantly upregulated and 143 genes downregulated following HP exposure. We annotated HP-responsive operons and all transcriptional start sites (TSSs) and identified which TSSs responded to HP treatment. We compared the HP responses and other previously reported genes and found only partial overlapping of other regulatory networks, indicating that the response to HP involves multiple biological functions. Using a representative subset of responsive genes, we validated the RNA-Seq results and found that the HP transcriptome was similar to that of sublethal organic peroxide. None of the genes in the representative subset, however, responded to sublethal levels of HOCl or O2 -. These results support the idea that N. gonorrhoeae may use variations in HP levels as a signal for different stages of infection. IMPORTANCE The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. This bacterium encounters hydrogen peroxide produced from host cells during infection, but the organism survives in the presence of this antimicrobial agent. This work shows that the bacterium responds to hydrogen peroxide by regulating the expression of many genes involved in multiple processes.

 

Structure of the Recombinant Neisseria gonorrhoeae Adhesin Complex Protein (rNg-ACP) and Generation of Murine Antibodies with Bactericidal Activity against Gonococci.

Almonacid-Mendoza HL, Humbert MV, Dijokaite A, Cleary DW, Soo Y, Hung MC, Orr CM, Machelett MM, Tews I, Christodoulides M.  (Full Text)

mSphere. 2018 Oct 10;3(5). pii: e00331-18. doi: 10.1128/mSphere.00331-18.

Abstract

Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and no effective vaccine exists currently. In this study, the structure, biological properties, and vaccine potential of the Ng-adhesin complex protein (Ng-ACP) are presented. The crystal structure of recombinant Ng-ACP (rNg-ACP) protein was solved at 1.65 Å. Diversity and conservation of Ng-ACP were examined in different Neisseria species and gonococcal isolates (https://pubmlst.org/neisseria/ database) in silico, and protein expression among 50 gonococcal strains in the Centers for Disease Control and Prevention/Food and Drug Administration (CDCP/FDA) AR Isolate Bank was examined by Western blotting. Murine antisera were raised to allele 10 (strain P9-17)-encoded rNg-ACP protein with different adjuvants and examined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and a human serum bactericidal assay. Rabbit antiserum to rNg-ACP was tested for its ability to prevent Ng-ACP from inhibiting human lysozyme activity in vitro. Ng-ACP is structurally homologous to Neisseria meningitidis ACP and MliC/PliC lysozyme inhibitors. Gonococci expressed predominantly allele 10- and allele 6-encoded Ng-ACP (81% and 15% of isolates, respectively). Murine antisera were bactericidal (titers of 64 to 512, P<0.05) for the homologous P9-17 strain and heterologous (allele 6) FA1090 strain. Rabbit anti-rNg-ACP serum prevented Ng-ACP from inhibiting human lysozyme with ∼100% efficiency. Ng-ACP protein was expressed by all 50 gonococcal isolates examined with minor differences in the relative levels of expression. rNg-ACP is a potential vaccine candidate that induces antibodies that (i) are bactericidal and (ii) prevent the gonococcus from inhibiting the lytic activity of an innate defense molecule.IMPORTANCE Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and the organism is listed by the World Health Organization as a high-priority pathogen for research and development of new control measures, including vaccines. In this study, we demonstrated that the N. gonorrhoeae adhesin complex protein (Ng-ACP) was conserved and expressed by 50 gonococcal strains and that recombinant proteins induced antibodies in mice that killed the bacteria in vitro We determined the structure of Ng-ACP by X-ray crystallography and investigated structural conservation with Neisseria meningitidis ACP and MliC/PliC proteins from other bacteria which act as inhibitors of the human innate defense molecule lysozyme. These findings are important and suggest that Ng-ACP could provide a potential dual target for tackling gonococcal infections.

 

Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus.

Rouquette-Loughlin CE, Reimche JL, Balthazar JT, Dhulipala V, Gernert KM, Kersh EN, Pham CD, Pettus K, Abrams AJ, Trees DL, St Cyr S, Shafer WM. (Full Text)

MBio. 2018 Nov 27;9(6). pii: e02281-18. doi: 10.1128/mBio.02281-18.

Abstract

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.