Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES - March, 2019

Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES

March, 2019

In the news



Why Sex-Related Infections Are Spreading Again

Gale J.  

Washington Post. 2019 

Sexually transmitted infections (STIs) are rebounding in developed countries after being firmly in retreat for decades. The consequences can be devastating. Syphilis, for example, causes more than 200,000 stillbirths and infant deaths worldwide annually, and years later can lead to blindness, dementia or paralysis. The resurgence is a result of multiple factors including inconsistent condom usage and the abuse of illicit recreational drugs. At the same time, some common STIs, such as gonorrhea and shigellosis, are becoming harder to treat due to antibiotic resistance. In the U.S., which has the highest rates of sexually transmitted disease in the developed world, the crisis is costing an estimated $16 billion annually in preventable health-care expenses.

Shield Diagnostics announces launch of Target-NG test for antibiotic susceptibility in Neisseria gonorrhoeae

Newswise. 2019 

SAN JOSE, Calif., March 14, 2019 /PRNewswire/ -- Shield Diagnostics, an Andreessen Horowitz-backed clinical laboratory tackling antibiotic resistance by bringing precision medicine to infectious disease, announced the launch of Target-NG, a rapid molecular test for antibiotic susceptibility in Neisseria gonorrhoeae.

 

Epidemiology and Surveillance

Genome-based epidemiology and antimicrobial resistance determinants of Neisseria gonorrhoeae isolates with decreased susceptibility and resistance to extended-spectrum cephalosporins in Argentina in 2011-16

Gianecini RA, Golparian D, Zittermann S, Litvik A, Gonzalez S, Oviedo C, Melano RG, Unemo M, Galarza P; Gonococcal Antimicrobial Susceptibility Surveillance Programme-Argentina (GASSP-AR) Working Group. (Full Text)

J Antimicrob Chemother. 2019 Feb 28. pii: dkz054. doi: 10.1093/jac/dkz054. [Epub ahead of print]

Abstract

OBJECTIVES:

Our aim was to describe the molecular epidemiology and antimicrobial resistance determinants of isolates of Neisseria gonorrhoeae with decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) in Argentina in 2011-16.



METHODS:

Gonococcal isolates (n = 158) with decreased susceptibility and resistance to ESCs collected in 2011-16 across Argentina were subjected to WGS and antimicrobial susceptibility testing for six antimicrobials.



RESULTS:

In total, 50% of the isolates were resistant to cefixime, 1.9% were resistant to ceftriaxone, 37.3% were resistant to azithromycin and 63.9% of the isolates showed an MDR phenotype. Resistance and decreased susceptibility to ESCs was mainly associated with isolates possessing the mosaic penA-34.001, in combination with an mtrR promoter A deletion, and PorB1b amino acid substitutions G120K/A121N. Phylogenetic analysis revealed two main clades of circulating strains, which were associated with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 and closely related STs, and characterized by a high prevalence rate, wide geographical distribution and temporal persistence.



CONCLUSIONS:

N. gonorrhoeae isolates with decreased susceptibility and resistance to ESCs in Argentina have emerged and rapidly spread mainly due to two clonal expansions after importation of one or two strains, which are associated with the international MDR NG-MAST ST1407 clone. The identification of the geographical dissemination and characteristics of these predominant clones may help to focus action plans and public health policies to control the spread of ESC resistance in Argentina. Dual antimicrobial therapy (ceftriaxone plus azithromycin) for gonorrhoea needs to be considered in Argentina.

Genetic relatedness of ceftriaxone-resistant and high-level azithromycin resistant Neisseria gonorrhoeae cases, United Kingdom and Australia, February to April 2018.

Jennison AV, Whiley D, Lahra MM, Graham RM, Cole MJ, Hughes G, Fifer H, Andersson M, Edwards A, Eyre D. (Full Text)

Euro Surveill. 2019 Feb;24(8). doi: 10.2807/1560-7917.ES.2019.24.8.1900118. 

Abstract

Between February and April 2018, three ceftriaxone-resistant and high-level azithromycin-resistant Neisseria gonorrhoeae cases were identified; one in the United Kingdom and two in Australia. Whole genome sequencing was used to show that the isolates from these cases belong to a single gonococcal clone, which we name the A2543 clone.

Evidence for Clonally Associated Increasing Rates of Azithromycin Resistant Neisseria gonorrhoeae in Rio de Janeiro, Brazil.

Barros Dos Santos KT, Skaf LB, Justo-da-Silva LH, Medeiros RC, Francisco Junior RDS, Caniné MCA, Fracalanzza SEL, Bonelli RR. (Full Text)

Biomed Res Int. 2019 Jan 20;2019:3180580. doi: 10.1155/2019/3180580. eCollection 2019.

Abstract

OBJECTIVES:

Azithromycin is one of the drugs used in the combined therapy for syndromic treatment of gonorrhoea in many countries, including Brazil. Our research group, which receives isolates from clinical laboratories since 2006, has detected, after 2016, a tendency of rising rates of azithromycin resistance, with isolates showing higher minimal inhibitory concentrations (MICs) than those previously reported in this country. In this study, we report the susceptibility to azithromycin of 93 N. gonorrhoeae isolates obtained between 2014 and 2017. Strains with MIC ≥2 μg/mL were characterized according to azithromycin resistance mechanisms and strain typing. Results indicate that azithromycin resistance has emerged in all these years in unrelated MLST-STs, but after 2016 a clonal complex connected with ST1901 has been more frequently detected, grouping isolates with MIC varying from 2 to 64 μg/mL, with DelA mutations at the mtrR promoter region associated or not with mutations at rrl alleles. High rates of azithromycin resistance may compromise the use of this drug in the combined therapy with ceftriaxone. Inclusion of Rio de Janeiro in the Brazilian gonococcal surveillance program is important to evaluate if this data indicates an epidemiological phenomenon in the country.

Emergence and Spread of Cephalosporin-Resistant Neisseria gonorrhoeae with Mosaic penA Alleles, South Korea, 2012-2017.

Lee H, Suh YH, Lee S, Kim YK, Han MS, Bae HG, Unemo M, Lee K. (Full Text)

Emerg Infect Dis. 2019 Mar;25(3):416-424. doi: 10.3201/eid2503.181503.

Abstract

In South Korea, surveillance of antimicrobial drug resistance in Neisseria gonorrhoeae is extremely limited. We describe the emergence and subsequent national spread of N. gonorrhoeae strains with mosaic penA alleles associated with decreased susceptibility and resistance to extended-spectrum cephalosporins. From 2012 through 2017, the proportion of mosaic penA alleles in gonococcal-positive nucleic acid amplification test (NAAT) specimens across South Korea increased from 1.1% to 23.9%. Gonococcal strains with mosaic penA alleles emerged in the international hubs of Seoul in Gyeonggi Province and Busan in South Gyeongsang Province and subsequently spread across South Korea. Most common was mosaic penA-10.001 (n = 572 isolates; 94.7%), which is associated with cefixime resistance. We also identified mosaic penA-34.001 and penA-60.001, both of which are associated with multidrug-resistant gonococcal strains and spread of cefixime and ceftriaxone resistance. Implementation of molecular resistance prediction from N. gonorrhoeae-positive nucleic acid amplification test specimens is imperative in South Korea and internationally.

Evidence of Recent Genomic Evolution in Gonococcal Strains with Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014-2016.

Thomas JC, Seby S, Abrams AJ, Cartee J, Lucking S, Vidyaprakash E, Schmerer M, Pham CD, Hong J, Torrone E, St Cyr S, Shafer WM, Bernstein K, Kersh EN, Gernert KM; Antimicrobial-Resistant Neisseria gonorrhoeae Working Group. (Full Text)

J Infect Dis. 2019 Feb 21. pii: jiz079. doi: 10.1093/infdis/jiz079. [Epub ahead of print]

Abstract

BACKGROUND:

Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States from 2014 to 2016.



METHODS:

We sequenced the genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project (GISP). We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations.



RESULTS:

We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin MICs (AZI em), characterized by a mosaic mtr locus (C-substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZI em strains. Another clonal group (MLST ST1901) possessed seven unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance.



CONCLUSIONS:

Whole genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection within a patient.

 

Novel detection strategies and diagnostics

Closed Genome Sequences of Clinical Neisseria gonorrhoeae Strains Obtained from Combined Oxford Nanopore and Illumina Sequencing.

White RC, Torralba M, Colt K, Harrison F, Goglin K, Nguyen K, D'Souza R, Bristow CC, Ellis O, Soge OO, Klausner JD, Fouts DE. (Full Text)

Microbiol Resour Announc. 2019 Feb 28;8(9). pii: e00072-19. doi: 10.1128/MRA.00072-19. eCollection 2019 Feb. 

Abstract

Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common notifiable disease in the United States. Here, we used a hybrid approach combining Oxford Nanopore Technologies MinION and Illumina MiSeq sequencing data to obtain closed genome sequences of nine clinical N. gonorrhoeae isolates.

Gen2Epi: an automated whole-genome sequencing pipeline for linking full genomes to antimicrobial susceptibility and molecular epidemiological data in Neisseria gonorrhoeae

Singh R, Dillon JR, Demczuk W, Kusalik A. (Full Text)

BMC Genomics. 2019 Mar 4;20(1):165. doi: 10.1186/s12864-019-5542-3.

Abstract
BACKGROUND:

Recent advances in whole genome sequencing (WGS) based technologies have facilitated multi-step applications for predicting antimicrobial resistance (AMR) and investigating the molecular epidemiology of Neisseria gonorrhoeae. However, generating full scaffolds of N. gonorrhoeae genomes from short reads, and the assignment of molecular epidemiological information (NG-MLST, NG-MAST, and NG-STAR) to multiple assembled samples, is challenging due to required manual tasks such as annotating antimicrobial resistance determinants with standard nomenclature for a large number of genomes.



RESULTS:

We present Gen2Epi, a pipeline that assembles short reads into full scaffolds and automatically assigns molecular epidemiological and AMR information to the assembled genomes. Gen2Epi is a command-line tool integrating third-party software and tailored specifically for N. gonorrhoeae. For its evaluation, the Gen2Epi pipeline successfully assembled the WGS short reads from 1484 N. gonorrhoeae samples into full-length genomes for both chromosomes and plasmids and was able to assign in silico molecular determinant information to each dataset automatically. The assemblies were generated using raw as well as trimmed short reads. The median genome coverage of full-length scaffolds and "N" statistics (N50, NG50, and NGA50) were higher than, or comparable to, previously published results and the scaffolding process improved the quality of the draft genome assemblies. Molecular antimicrobial resistant (AMR) determinants identified by Gen2Epi reproduced information for the 1484 samples as previously reported, including NG-MLST, NG-MAST, and NG-STAR molecular sequence types.



CONCLUSIONS:

Gen2Epi can be used to assemble short reads into full-length genomes and assign accurate molecular marker and AMR information automatically from NG-STAR, NG-MAST, and NG-MLST. Gen2Epi is publicly available under "CC BY-NC 2.0 CA" Creative Commons licensing as a VirtualBox image containing the constituent software components running on the LINUX operating system (CentOS 7). The image and associated documentation are available via anonymous FTP at ftp://www.cs.usask.ca/pub/combi or ftp://ftp.cs.usask.ca/pub/combi..

A Real-Time PCR Assay for the Detection of a penA Mutation Associated with Ceftriaxone Resistance in Neisseria gonorrhoeae.

Shimuta K, Igawa G, Yasuda M, Deguchi T, Nakayama SI, Ohnishi M. (Full Text)

J Glob Antimicrob Resist. 2019 Feb 27. pii: S2213-7165(19)30051-7. doi: 10.1016/j.jgar.2019.02.011. [Epub ahead of print]

Abstract

OBJECTIVES:

Ceftriaxone (CRO) resistance is spreading worldwide, hindering the effective treatment of gonococcal infections. In this study, a detection system for the genomic DNA of CRO-resistant Neisseria gonorrhoeae strains was developed to improve the surveillance of antimicrobial resistance.



METHODS:

We designed a real-time PCR assay targeting the penA gene of CRO-resistant Neisseria gonorrhoeae strains recently isolated. Primer and probe sequence information was obtained from sequence comparisons between penA of Neisseria spp. and penA of CRO-resistant N. gonorrhoeae strains.



RESULTS:

Using our assay, a positive reaction was observed using the genomic DNA of three strains (GU140106, FC428, and A8806). The assay was evaluated using genomic DNA of 204 N. gonorrhoeae and 95 Neisseria spp. isolates with known minimum inhibitory concentrations of CRO. Following PCR assays for these strains, we positively identified three FC428-related strains, which possessed penA-60.001, whereas the remaining 201 N. gonorrhoeae strains and 95 Neisseria spp. strains were negative.



CONCLUSIONS:

We designed a real-time PCR-based assay to detect the genomic DNA of strains harboring mosaic penA-59.001 (GU140106), penA-60.001 (FC428), and penA-64.001 (A8806) alleles and to discriminate them from N. gonorrhoeae and Neisseria spp. strains harboring other genes.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

Wang LC, Wagner J, Capino A, Nesbit E, Song W, Stein DC. (Full Text)

J Vis Exp. 2019 Feb 8;(144). doi: 10.3791/58978.

Abstract

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.
 

 

Novel antibiotics, mechanisms, vaccine, and other advances

Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production.

Baarda BI, Zielke RA, Le Van A, Jerse AE, Sikora AE. 

PLoS Pathog. 2019 Mar 7;15(3):e1007385. doi: 10.1371/journal.ppat.1007385. [Epub ahead of print]

Abstract

The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence.

 

Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species.

Fegan JE, Calmettes C, Islam EA, Ahn SK, Chaudhuri S, Yu RH, Gray-Owen SD, Moraes TF, Schryvers AB. (Full Text)

Front Immunol. 2019 Feb 19;10:247. doi: 10.3389/fimmu.2019.00247. eCollection 2019.

Abstract

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.

The Neisseria gonorrhoeae Methionine Sulfoxide Reductase (MsrA/B) Is a Surface Exposed, Immunogenic, Vaccine Candidate.

Jen FE, Semchenko EA, Day CJ, Seib KL, Jennings MP. (Full Text)

Front Immunol. 2019 Feb 6;10:137. doi: 10.3389/fimmu.2019.00137. eCollection 2019.

Abstract

Control of the sexually transmitted infection gonorrhea is a major public health challenge, due to the recent emergence of multidrug resistant strains of Neisseria gonorrhoeae, and there is an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study, we evaluated the methionine sulfoxide reductase (MsrA/B) of N. gonorrhoeae as a potential vaccine candidate, in terms of its expression, sequence conservation, localization, immunogenicity, and the functional activity of antibodies raised to it. Gonococcal MsrA/B has previously been shown to reduce methionine sulfoxide [Met(O)] to methionine (Met) in oxidized proteins and protect against oxidative stress. Here we have shown that the gene encoding MsrA/B is present, highly conserved, and expressed in all N. gonorrhoeae strains investigated, and we determined that MsrA/B is surface is exposed on N. gonorrhoeae. Recombinant MsrA/B is immunogenic, and mice immunized with MsrA/B and either aluminum hydroxide gel adjuvant or Freund's adjuvant generated a humoral immune response, with predominantly IgG1 antibodies. Higher titers of IgG2a, IgG2b, and IgG3 were detected in mice immunized with MsrA/B-Freund's adjuvant compared to MsrA/B-aluminum hydroxide adjuvant, while IgM titers were similar for both adjuvants. Antibodies generated by MsrA/B-Freund's in mice mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity. Anti-MsrA/B was also able to functionally block the activity of MsrA/B by inhibiting binding to its substrate, Met(O). We propose that recombinant MsrA/B is a promising vaccine antigen for N. gonorrhoeae.



In vitro activity of the novel oral antimicrobial SMT-571, with a new mechanism of action, against MDR and XDR Neisseria gonorrhoeae: future treatment option for gonorrhoea?

Jacobsson S, Mason C, Khan N, Meo P, Unemo M. (Full Text)

J Antimicrob Chemother. 2019 Feb 18. pii: dkz060. doi: 10.1093/jac/dkz060. [Epub ahead of print]

Abstract

BACKGROUND:

Lack of effective treatment of gonorrhoea due to increasing antimicrobial resistance in Neisseria gonorrhoeae is a serious threat to the management and control of the infection. Novel antimicrobials are required to prevent the infection becoming untreatable.



OBJECTIVES:

Herein, we investigated the in vitro activity of a novel small-molecule antimicrobial with a new mechanism of action, SMT-571, against a large collection of clinical N. gonorrhoeae isolates (n = 228) and international gonococcal reference strains (n = 34), including numerous MDR and XDR gonococcal isolates.



METHODS:

MICs of SMT-571 were determined by agar dilution and MICs of ceftriaxone, cefixime, azithromycin, ciprofloxacin, ampicillin, spectinomycin and tetracycline were determined by Etest.



RESULTS:

SMT-571 showed potent in vitro activity against all the tested N. gonorrhoeae isolates (n = 262). The MICs ranged from 0.064 to 0.125 mg/L and the MIC50, MIC90 and modal MIC were all 0.125 mg/L. No cross-resistance or correlation between the MICs of SMT-571 and comparator agents was seen.



CONCLUSIONS:

SMT-571 demonstrated potent in vitro activity against all tested gonococcal isolates and no cross-resistance to previously and currently used antimicrobials was seen. With its promising supplementary in vitro and in vivo preclinical data, including high levels of oral bioavailability, SMT-571 could be an effective option for the oral treatment of gonorrhoea. Randomized controlled clinical trials for gonorrhoea that examine the treatment efficacy, pharmacokinetics/pharmacodynamics, toxicity and safety of SMT-571, and include urogenital and extragenital (rectal and pharyngeal) samples, are crucial.