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Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Special Issue: Antimicrobial Therapeutics Reviews REVIEW Applications of genomics to slow the spread of multidrug-resistant gonorrhoeae

Tatum D. Mortimer 1 and Yonatan H. Grad 1,2 1Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts. 2Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Address for correspondence: Yonatan H. Grad, Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, 665 Huntington Ave., Building 1, Room 715, Boston, MA 02115. [email protected]

Infections with , a sexually transmitted that causes urethritis, cervicitis, and more severe complications, are increasing. is typically treated with ; however, N. gonorrhoeae has rapidly acquired resistance to many classes, and lineages with reduced susceptibility to the currently recommended therapies are emerging worldwide. In this review, we discuss the contributions of whole genome sequencing (WGS) to our understanding of resistant N. gonorrhoeae. Genomics has illuminated the evolutionary origins and population structure of N. gonorrhoeae and the magnitude of horizontal gene transfer within and between Neisseria species. WGS can be used to predict the susceptibility of N. gonorrhoeae based on known resistance determinants, track the spread of these determinants throughout the N. gonorrhoeae population, and identify novel loci contributing to resistance. WGS has also allowed more detailed epidemiological analysis of transmission of N. gonorrhoeae between individuals and populations than previously used typing methods. Ongoing N. gonorrhoeae genomics will complement other laboratory techniques to understand the biology and evolution of the pathogen, improve diagnostics and treatment in the clinic, and inform public health policies to limit the impact of antibiotic resistance.

Keywords: gonorrhea; whole genome sequencing; antibiotic resistance; epidemiology

Introduction ceftriaxone and azithromycin is currently recom- mended based on the hypothesis that the two drugs Gonorrhea, caused by Neisseria gonorrhoeae, is the together will help to curb the spread of resistant second most common sexually transmitted infec- lineages.4 With the dearth of antibiotic options for tion with 468,514 reported cases in the United States gonorrhea and concern for rising levels of resistance in 20161 and an estimated 78 million cases world- in this highly prevalent infection, the Centers for wide annually.2 N. gonorrhoeae infection rates in Disease Control and Prevention (CDC) highlighted the United States have increased yearly since 2013, N. gonorrhoeae resistant to cephalosporins as one and rate increases are larger in men than women.1 of three urgent threats among antibiotic resistant N. gonorrhoeae is particularly prevalent in men who .5 have sex with men (MSM), and 37.8% of isolates The World Health Organization (WHO) has were collected from MSM during surveillance in identified several strategies to control the spread the United States in 2016.3 of antibiotic resistant N. gonorrhoeae, centering Bacterial are becoming increasingly on improved methods for diagnosis, strength- resistant to antibiotics, and N. gonorrhoeae is no ened detection and surveillance of resistance, and exception. N. gonorrhoeae has developed resistance identification of new treatment strategies.2 Cur- to every antibiotic used for treatment through a vari- rent challenges include the rapid identification of ety of mechanisms (Table 1), and dual therapy with gonococcal infection and antibiotic susceptibility, doi: 10.1111/nyas.13871

Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. 1 Genomics of multidrug-resistant Neisseria gonorrhoeae Mortimer & Grad

Table 1. Mechanisms of antibiotic resistance in Neisseria gonorrhoeae

MIC breakpoints137 Drug (␮g/mL) S, R Gene (codon/allele) Mechanism Year

Sulfonamides folP (R228)138,139 TM 2005 Penicillin ࣘ0.06, ࣙ2 penA45,140 TM 1975 (PEN) porB (G120K, A121D/N)140,141 P1975 mtrR (A39T, G45D, 1 bp del in promoter)140,142–144 E 1975 61 blaTEM I 1976 ponA (L421P)87 TM 2002 pilQ (E666K)87,88 P2002 Tetracycline ࣘ0.25, ࣙ2 porB (G120K, A121D/N)140,141 P 1975 (TET) mtrR (A39T, G45D, 1 bp del in promoter)140,142–144 E 1975 tetM32 TM 1986 pilQ (E666K)87,88 P 2002 rpsJ (V57M)145,146 TM 1974 Ciprofloxacin ࣘ0.06, ࣙ1 gyrA (S91F, D95A/N/G)147,148 TM 1994 (CIP) parC (D86N, S87R/N, S88P, E91K)147,149 TM 1994 parE (G410V)148 TM 2002 norM (-35 promoter sequence, RBS)150 E 2003 Spectinomycin ࣘ32, ࣙ128 rpsE (T24P, deletion V27, A82G)151,152 TM 2013 16S rDNA (C1187)153 TM 2000 Azithromycin mtrR (A39T, G45D, 1 bp del in promoter) 140,142–144 E 1975 (AZI) mosaic mtr operon47 E 2016 ermBCF 57 I 1999 23S rDNA (C2611T, A2059G)154,155 TM 2002 mef156 E 2000 macAB (-10 promoter sequence)150 E 2003 rplV (3 tandem duplications)22 TM 2016 rplD (G68, G70)22 TM 2016 Cefixime ࣘ0.25, – mosaic penA46,157 TM 2002 (CFX) Ceftriaxone ࣘ0.25a,– mosaicpenA157 TM 2005 (CRO) porB158 P2009 mtrR158 E 2009

TM, target modification; P, permeability; E, efflux; I, inactivation. aReduced susceptibility to CRO is also described as ࣙ0.125 ␮g/mL. determination of transmission links between indi- (as of December 10, 2017). Here, we review the viduals and populations, and tailoring treatment background on gonorrhea, resistant gonococcus, and intervention strategies to optimally slow or con- and the ways in which researchers are exploring how tain the spread of antibiotic resistance (Box 1). to integrate these data to advance clinical care and Whole genome sequencing (WGS), along with public health management of gonorrhea. other methodological and technical innovations, will enable new approaches to address each of Neisseria gonorrhoeae: clinical these challenges. WGS can be used to iden- manifestations and origins tify outbreaks,6 enhance diagnostics by pathogen The Gram negative diplococcus N. gonorrhoeae is and antibiotic resistance detection,7–9 and perform a major cause of urethritis as well as of cervicitis pathogen surveillance.10 Over the 20 years since the and pelvic inflammatory disease.11 N. gonorrhoeae first gonococcal genome was sequenced, 15 finished also causes disease outside of the urogenital tract, genomes, 413 draft genome assemblies, and 4795 including pharyngeal and rectal infection, which sequencing runs have been made publicly available tend to be asymptomatic, as well as conjunctivitis,

2 Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. Mortimer & Grad Genomics of multidrug-resistant Neisseria gonorrhoeae

Box 1: Current challenges and contributions of WGS at different scales Neisseria gonorrhoeae populations r r Identification of novel resistance and compensatory mutations r Characterize selective pressures and fitness costs r Identify targets for vaccines r Frequency of antimicrobial resistance (AMR) acquisition and interspecies mosaicism Roles of major and minor within-host populations in transmission and susceptibility

In the clinic r r Rapidly identify infection and colonization r Identify susceptibility of strains and appropriate antibiotic treatment Identify clinically relevant mixed infections

Local transmission networks r r Identify links among individuals r Tailor antibiotic use at local scale Translation to local policies and interventions

Global scales r r Identify links among populations r Tailor antibiotic use at global scale Translation to global policies and interventions

disseminated gonococcal infection with associated as other Neisseria (Fig. 1), suggesting the species mono- and oligoarticular arthritis, and Fitz–Hugh– has arisen relatively recently18–21 and likely as an Curtis syndrome (perihepatitis).12 Antibiotic offshoot from meningococcus. These observations therapy cures gonococcal infections and is indi- invite speculation about the origins and ongoing cated for both symptomatic and asymptomatic evolution of Neisseria, raising the possibility that infections. contact between the oropharynx and other mucosa N. gonorrhoeae is a member of the Neisseria presents opportunities for Neisseria to adapt to and genus, with most other species considered com- inhabit new niches, and the confluence of the right mensal oropharyngeal flora. The clinical manifes- genetic and behavior contexts to promote trans- tations of N. gonorrhoeae lead to the definition mission may facilitate the emergence of Neisseria of gonococcus along with strains. (the meningococcus)—the cause of meningococcal Population structure analyses based on Bayesian meningitis and septicemia—as the pathogenic Neis- methods have identified 5–12 clusters in N. gon- seria (Fig. 1).13 The standard pigeonholing of these orrhoeae populations, depending on the data set, species—while a convenient shorthand—belies the and these clusters and major clades defined by clinical observations that not all the Gram negative phylogenetic analysis have little to no geographic diplococci that cause “gonorrhea-like” symptoms structure.21–24 In N. meningitidis, clade-specific are N. gonorrhoeae. Meningococcus and Neisseria restriction modification systems, competition lactamica have been isolated in urethritis cases,14 between lineages, and selection by the immune sys- and a recent outbreak of meningococcal urethri- tem contribute to the population structure,25,26 but tis renewed interest in the question of whether the factors that maintain population structure in N. adaptation to the urogenital niche may happen gonorrhoeae have not been identified. The global periodically.15–17 N. gonorrhoeae is not as diverse distribution of these major groups suggests that

Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. 3 Genomics of multidrug-resistant Neisseria gonorrhoeae Mortimer & Grad

N. gonorrhoeae

N. meningitidis

N. lactamica

0.06

Figure 1. Phylogeny of Neisseria. All publicly available genomes from the Neisseria genus were downloaded from NCBI (as of December 30, 2017) and annotated with Prokka v 1.12.134 N. gonorrhoeae and N. meningitidis were subsampled to 20 genomes. Core genes (n = 236) were identified and aligned with Roary v 3.12.0.135 Approximate maximum likelihood phylogenetic analysis was performed using FastTree v 2.1.9.136 The phylogeny was visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Branches are scaled by substitutions per site. Data are available at https://doi.org/10.6084/m9.figshare.5877486. international transmission of N. gonorrhoeae is rel- cervical infection may yield a relatively low pharyn- atively common. geal antibiotic concentration inadequate for eradi- cation and thereby promote resistance emergence. A Acquisition of antibiotic resistance recent epidemiological study relating annual antibi- Antibiotic exposure drives the emergence of resis- otic consumption and gonococcal resistance sug- tance in gonococcus. These exposures derive from gested that the pressure from bystander selection treatment for gonorrhea and potentially through may be small to none,27 whereas a study from “bystander effect,” during treatment for other infec- the Netherlands indicated that previous exposure tions in individuals who are asymptomatically to azithromycin is associated with higher levels of infected with gonococcus. The variable tissue pen- azithromycin resistance, perhaps reflecting the long etration of antibiotics into the pharynx, a site of half- of azithromycin and the observation that gonococcal infections, is one likely source of resis- individuals previously infected with gonorrhea are tance, as the dose needed to clear urethral or at higher risk for subsequent infection.28

4 Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. Mortimer & Grad Genomics of multidrug-resistant Neisseria gonorrhoeae

In addition to de novo acquisition of resistance will uptake genetic material from other Neisseria, due to antibiotic exposure, genes and alleles can eas- including N. meningitidis, N. lactamica, and other ily move between N. gonorrhoeae lineages, provid- commensals,44 further implicating the orophar- ing another mechanism for acquisition of antibiotic ynx as a potential site of resistance acquisition. resistance. N. gonorrhoeae harbors several mobile Mosaicism with other Neisseria species has also been genetic elements. Almost all gonococcal isolates an important source for the acquisition of antibi- encode a small, cryptic plasmid, which can also be otic resistance in N. gonorrhoeae. Most notably, integrated into the chromosome.29,30 Aconjugative mosaic sequences in penA have resulted in resis- plasmid can also be found, and a transposon encod- tance to penicillin and reduced susceptibility to ing tetM, which mediates tetracycline resistance, has extended spectrum cephalosporins (ESCs),45,46 and inserted into this plasmid and mobilized among amosaicmtr operon has been shown to be asso- N. gonorrhoeae (Dutch type, so-called because the ciated with azithromycin resistance in the United initially described plasmid of this type was encoded States22,47 and Australia.48,49 In a dataset of N. by an isolate from the Netherlands),31,32 A dis- gonorrhoeae isolated in the United States, 6% of tinct tetM determinant can be found on another recombinant tracts matched sequences found in plasmid backbone (American type, as it was first other Neisseria, including an acquisition of tbpB described in an isolate from the United States),31,32 from N. meningitidis.50 Multiple N. gonorrhoeae suggesting two introductions of tetM-mediated lineages have acquired porA sequences from N. resistance into the N. gonorrhoeae population.31,33 meningitidis, replacing the gonococcal porA pseu- 51 A diverse set of plasmids encoding blaTEM beta lac- dogene that is occasionally used for diagnostics. tamase is transmitted among N. gonorrhoeae pop- Exchange among Neisseria species is not unidirec- ulations; however, they are all derivatives of the tional, and urethritis-associated N. meningitidis iso- Asia-type plasmid arising by repeat-mediated lates contain recombinant tracts originating from deletions or duplications.34 This plasmid likely N. gonorrhoeae.17 originated in species after a transpo- Despite being a potential reservoir of resistance son insertion into a cryptic plasmid and was hor- determinants for pathogenic species, the prevalence izontally transferred to N. gonorrhoeae.35 Finally, and mechanisms of antibiotic resistance in com- many N. gonorrhoeae contain the gonococcal genetic mensal Neisseria are largely unknown. Interspecies island (GGI),36,37 which encodes machinery for exchange of penA sequences has long been recog- a Type IV secretion system (T4SS) that secretes nized as contributing to penicillin resistance in both DNA into the environment, as well as proteins of pathogenic and commensal Neisseria.52–54 Trans- unknown function.38 Similar T4SSs can be found formation of N. gonorrhoeae with penA sequences in other ; however, flanking regions from a ceftriaxone resistant isolate are not conserved.39 DNA secretion may facilitate resulted in transformants with minimum inhibitory horizontal gene transfer, including resistance acqui- concentrations (MICs) to ESCs similar to the donor sition, and biofilm formation.36,37,40 The T4SS can and a mosaic penA similar to alleles found in clin- also enable intracellular survival of N. gonorrhoeae ical N. gonorrhoeae isolates with high-level cef- via iron acquisition by an unknown mechanism, triaxone resistance.55 It is unknown if the penA which is independent of DNA secretion.41 These sequences originated in N. cinerea or were horizon- mobile elements are found in diverse N. gonorrhoeae tally transferred to both N. cinerea and N. gonor- strains and are not confined to a particular clade. rhoeae from an unknown source. The only survey of Besides the cryptic plasmid, plasmids and the GGI commensal Neisseria resistance to ESCs showed that are found at intermediate frequencies in the N. gon- 93% and 100% of Neisseria subflava samples from orrhoeae population, suggesting that a fitness cost Japan were susceptible to cefixime and ceftriaxone, to carrying these mobile elements exists in some respectively.56 Resistance to penicillin, tetracycline, niches. and ciprofloxacin was also identified; however, the Neisseria species are naturally competent,42 with genetic mechanisms of resistance were not deter- a preference for DNA carrying a DNA uptake mined. Commensal Neisseria have also been shown sequence.43 In addition to exchanging alleles within to encode erm genes, which mediate macrolide the N. gonorrhoeae population, N. gonorrhoeae resistance.57

Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. 5 Genomics of multidrug-resistant Neisseria gonorrhoeae Mortimer & Grad

From pathogen to superbug: treatment Surveillance Network. Similarly, the European and resistance Gonococcal Antibiotic Surveillance Programme N. gonorrhoeae has routinely developed resistance to (GASP) monitors rates of gonorrhea and antibiotic each of the antibiotics used to treat it.58 Resistance to resistance in 27 European countries. There are also sulfonamides and low-level resistance to penicillin active surveillance efforts in China (China GASP), emerged in the 1940s.59,60 Penicillin continued to Australia (Australia GSP), and the UK (GRASP,Sex- be the most common treatment for gonorrhea for ually Transmitted Bacteria Reference Unit). In a several decades despite slowly increasing resistance partnership between CDC and WHO, the Enhanced due to chromosomal mutations. High-level resis- Gonorrhea Antimicrobial Surveillance Project was tance to penicillin via plasmid and chromosomally initiated in 2015 to monitor antibiotic resistance 76 mediated mechanisms was reported in 1976 and worldwide, particularly in high-risk populations. 1986, respectively.61,62 Tetracycline resistance can Currently, surveillance data are particularly sparse 67 also be mediated by both determinants on plasmids in Central America, Africa, and Central Asia. or the chromosome, and N. gonorrhoeae harboring Since there is no point-of-care (POC) antibiotic tetM encoding plasmids was first described in susceptibility testing, antibiotic selection for gonor- 1986.32 Clinical trials to study the effectiveness rhea treatment is based on recommended drugs and of ciprofloxacin to treat gonorrhea began in doses per national or regional guidelines. A recent 1983,63 and, though it was not recommended as review of N. gonorrhoeae treatment guidelines shows therapy in the United States until 1993,64 reduced that organizations suggest 250–500 mg of ceftriax- susceptibility to ciprofloxacin had already been one intramuscularly and 1–2 g azithromycin orally reported as early as 1990.65 In 2007, with rapidly for the treatment of uncomplicated infections; 400– rising prevalence of resistance, the CDC recom- 800mgcefiximeorallyisprovidedasanalternative 77 mended that ciprofloxacin no longer be used for to ceftriaxone in WHO and Canadian guidelines. gonorrhea treatment.66 In Southeast Asia and the The WHO also recommends ceftriaxone single ther- Western Pacific, rates of ciprofloxacin resistance apy. However, this review was limited in scope to remain extremely high (>90% in 2014).67 Even guidelines published in English, and guidelines from as first-line antibiotic therapies have changed, Africa, Asia, and South America were not included resistance to older therapies remains high in N. beyond WHO recommendations. To limit empiric gonorrhoeae populations across the globe.68–72 treatment failures, it has been the practice to switch For example, 44.1% of isolates collected by the the recommended first-line antibiotics when preva- Gonococcal Isolate Surveillance Project (GISP) lence of resistance exceeds 5%, though it is worth in 2016 were resistant to penicillin, tetracycline, noting that more nuanced strategies for optimizing and/or ciprofloxacin.1 With the idea that dual antibiotic use are needed. therapy would be helpful to halt the development The clonality and number of times drug resis- of resistance to the remaining effective antibiotics, tant lineages have emerged varies by drug and the CDC currently recommends ceftriaxone and mechanism of resistance. Chromosomal mutations azithromycin dual therapy.73 However, resistance mediating low-level resistance to beta-lactams and to azithromycin and ESCs is emerging,74 and treat- tetracycline are common in N. gonorrhoeae isolates, 22 ment failure with this regimen has been reported.75 and they are widespread across the phylogeny. Since susceptibility is not routinely measured in While azithromycin resistance is less common the clinic, the monitoring of antibiotic resistance than resistance to previously used drugs, resis- prevalence falls to surveillance programs, which tance or reduced susceptibility has arisen across 22,78,79 measure levels of resistance in the N. gonorrhoeae a large number of genomic backgrounds. population and inform treatment guidelines. In In contrast, reduced susceptibility to ESCs (ESC- the United States, the GISP collects the first 25 RS) is primarily clonal, and the majority of urethral isolates each month from sentinel clin- ESC-RS isolates belong to only a few major 22,50 ics for susceptibility testing. This project is being lineages. In isolates from the United States, enhanced (eGISP) to include isolates from addi- azithromycin reduced susceptibility (AZI-RS) is tional body sites and collect additional demographic estimated to have been acquired 75 times, and and behavioral data from patients through the STD reversion to susceptibility is also relatively frequent

6 Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. Mortimer & Grad Genomics of multidrug-resistant Neisseria gonorrhoeae

(42 times).22 In contrast, ciprofloxacin resistance reduced susceptibility to both azithromycin and has only been acquired 11 times, but there are far ESCs are circulating in Canada,89 and a cluster of more ciprofloxacin resistant isolates in the data set isolates with high-level resistance to azithromycin (594 versus 294 AZI-RS).22 and reduced susceptibility to ESCs was identified An impediment to the emergence of antibiotic in Hawaii, suggesting ongoing transmission of resistant bacteria is the fitness cost incurred by this lineage.90 Interestingly, these isolates were resistance in environments without antibiotic selec- more closely related to an isolate associated with tive pressure. Resistant bacteria have been shown to failed azithromycin and ceftriaxone dual therapy be less fit than their susceptible counterparts both in England75 than other contemporaneous isolates in vitro and in vivo for several bacterial species, from Hawaii. and resistant bacteria can mediate these costs by Azithromycin resistance and ESC reduced acquiring compensatory mutations.80 However, susceptibility have emerged on several genomic fitness costs of resistance and compensatory backgrounds. However, some clones are highly mutations in N. gonorrhoeae are understudied. The successful and have spread across the globe, while continued prevalence of penicillin, tetracycline, and others seem to result in few descendants or resis- ciprofloxacin resistant lineages despite discontinued tance is lost.22 One of the two major ESC-RS clades use of these antibiotics has been cited as evidence in the United States, for example, seems to have that there may be little to no fitness costs for ceased transmission after 2011.22 Some lineages these resistance determinants. Experimental work encoding azithromycin resistance are associated shows that mtr mutants have increased fitness in a with international transmission. For example, an mouse model.81,82 The most common mutations azithromycin resistant N. gonorrhoeae lineage has in gyrA conferring ciprofloxacin resistance also emerged in Scotland91 and spread to England do not have a cost in the mouse model, but the and Wales92 causing outbreaks.78 This lineage addition of parC mutations which appear in clinical has also been observed elsewhere in Europe79 and isolates does result in lower fitness.83 A modeling Australia.49 Whether success is attributable to muta- study by Whittles and colleagues using data on tions facilitating acquisition and maintenance of rates of cefixime susceptibility and cefixime use resistance determinants and sustained transmission for gonorrhea treatment in England suggests that remains unclear. Understanding the interactions cefixime resistance imparts a fitness cost compared between fitness costs of resistance, compensatory to susceptible lineages.84 Finally, reversion from mutations, and transmission of resistant lineages resistant to susceptible phenotypes has been is an important step in controlling the spread of observed in N. gonorrhoeae, which may indicate a antibiotic resistance in N. gonorrhoeae. fitness cost for resistance.22 Opportunities for addressing Interactions between resistance loci may also antimicrobial-resistant (AMR) gonorrhea: influence the emergence of multidrug resistant rapid diagnosis and antibiotic lineages. Resistance correlates across antibiotics, susceptibility assessment and many N. gonorrhoeae isolates are resistant to multiple drugs (17.6% of isolates collected by GISP The majority of gonorrhea cases are diagnosed via in 2016).85,86 Resistance determinants in N. gonor- a nucleic acid amplification test, through which N. rhoeae have been shown to have additive effects. For gonorrhoeae genetic material is detected in patient example, successive mutations in penA, mtr, porB, samples. Culture is rarely done, though it remains ponA, and pilQ contribute to high-level penicillin the only basis for assessing the MICs for antigono- resistance.62,87,88 Of particular concern currently coccal antibiotics. are lineages that have reduced susceptibility to both Gonorrhea is currently treated empirically based azithromycin and ESCs. In isolates sampled across on the extent of resistance described in surveil- the United States from 2000 to 2013, high-level AZI lance systems. While resistance to previously effec- resistance and ESC reduced susceptibility appeared tive antibiotics remains high in many areas of the to be anticorrelated, and depending on the genomic world, a large fraction of isolates remain suscepti- background, the presence of 23S rRNA mutations ble to older antibiotics and in some regions,68 most lowered MICs for other antibiotics.22 Isolates with isolates remain susceptible to all drugs. This has

Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. 7 Genomics of multidrug-resistant Neisseria gonorrhoeae Mortimer & Grad led to the idea that a POC diagnostic that detects reduced susceptibility to azithromycin could not antibiotic resistance—akin to the use of detec- be explained by known mutations.22 Continued tion of rpoB mutations to rapidly identify resistant surveillance and identification of novel resistance TB93,94—would increase treatment options, permit- mutations should be an ongoing effort. ting the use of antibiotics shelved because of concern Genomic epidemiology approaches to for the high fraction of empiric treatment failures. reducing the transmission of AMR A real-time PCR-based assay targeting gyrA gonorrhea has been shown to accurately detect ciprofloxacin resistance from patient samples (sensitivity: Prior to the advent of WGS, several typing schemes 95.8%, specificity: 100%).95 POCs have also been based on DNA sequences were developed to define developed for penicillin susceptibility; however, genetic relationships between N. gonorrhoeae accurate predictions require the assessment of mul- isolates and track the emergence and spread of tiple genetic loci.96,97 Modeling suggests that POC drug resistant gonorrhea. Multi-locus sequence testing for antibiotic resistance has the potential to typing (MLST), a technique used for many bacterial slow the spread of resistance;98 however, a test for species, uses the sequence of defined portions ciprofloxacin alone will not delay the increases in of a small number of genes (abcZ, adk, aroE, resistance to azithromycin and ESCs, whereas a POC fumC, gdh, pdhC,andpgm in Neisseria species) to for all three antibiotics would delay the rising levels assign an isolate to a sequence type.104 N. gonor- of resistance.99 rhoeae multiantigen sequence typing (NG-MAST), The increasingly portable and rapid turnaround another typing scheme, is based on the sequence of sequencing technologies may make sequencing of the hypervariable portions of porB and tbpB.105 in the clinic more practical. Just as nanopore Recently, targeted sequencing of known resistance technology has been used for real-time epidemi- determinants has also been utilized, and typing ological investigations for recent Ebola and Zika based on resistance profiles and housekeeping genes outbreaks,100,101 preliminary work has demon- has been proposed (NG-STAR).106 Online resources strated the feasibility of using the Oxford Nanopore for typing based on these schemes are available at MinION system as a gonococcal diagnostic, https://pubmlst.org/, http://www.ng-mast.net, and sequencing and predicting antibiotic susceptibility https://ngstar.canada.ca/. of N. gonorrhoeae isolates in less than 2 hours.102 Typing of bacterial strains has long been a com- One potential advantage of WGS in a diagnos- plement to typical epidemiological practices when tic test is that it can assay for multiple antibiotic investigating an outbreak. While WGS is generally resistance determinants at the same time. Several concordant with traditional typing methods like tools have been developed to predict antibiotic resis- MLST and NG-MAST,it can provide increased reso- tance from WGS data.8,9 Mykrobe8 and ARIBA9 lution and rule out potential transmission links that can identify resistance determinants directly from may be inferred by these methods.107 For exam- sequencing reads, eliminating the need for genome ple, in a study of N. gonorrhoeae specimens from assembly. Eyre and colleagues found that the MICs Brighton, UK, 8.8% of isolates collected within 28 for antibiotics commonly used to treat gonorrhea days of each other had the same NG-MAST type, but can be predicted from WGS data for 52% of isolates, only 3.6% of isolates were compatible with direct or and MICs within two dilutions of the observed MIC indirect transmission according to WGS results.24 In were predicted for 98% of isolates.103 British Columbia, monitoring of a database of HIV Ability to predict susceptibility profiles of clinical genotypes allowed for the identification of a grow- isolates is dependent on the comprehensiveness and ing transmission cluster and timely public health quality of resistance allele databases as well as under- follow-up;108 N. gonorrhoeae WGS may similarly standing of how loci interact to generate resistance. allow for identification of expanding lineages and Positive and negative predictive values of known prevention of further onward transmission. resistance alleles vary by antibiotic;37 for example, Since N. gonorrhoeae has a calculable substitution mutations associated with ciprofloxacin resistance rate at short genetic distances, the WGS data can are highly predictive, but in a recent WGS study be combined with dates of collection to infer likely of resistant isolates in the United States, 36% of transmission links.24 Moreover, the consistency of

8 Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. Mortimer & Grad Genomics of multidrug-resistant Neisseria gonorrhoeae estimates of the N. gonorrhoeae substitution rate spread of N. gonorrhoeae is common, which is also suggests that the transmissionnomogram calculated supported by the lack of geographic structure in N. byDeSilvaandcolleagues(basedonasubstitution gonorrhoeae populations.24 Frequent international rate of 3.55 single nucleotide polymorphisms per transmission of resistant strains poses a challenge to genomeperyear)couldbeapplicabletotransmis- local control efforts. sion of gonorrhea in other settings. Hypermutator Ongoing areas of research strains—which would have a faster substitution rate—have been observed in other pathogens, Even as we understand many of the most common including epidemic clones of N. meningitidis109 and genetic pathways to antibiotic resistance, our cata- antibiotic resistant aeruginosa,110 but log of resistance mechanisms and pathways remains gonococcal hypermutators have not been described. incomplete (Table 2). Genomic data provide a rich Combined with patient metadata, WGS can reveal source of information that can be used to define information about the demographics of contact the variants that cause resistance and their inter- networks. For example, clusters likely associated action with other loci in the genome. Bacterial with transmission in Brighton, UK contain iso- genome wide association studies (GWASs)113–115 lates from both HIV positive and HIV negative have successfully identified known and potentially patients,111 and clusters associated with heterosex- novel antibiotic resistance mutations in other bacte- ual women in Australia contain patients across a rial pathogens like tuberculosis and range of ages.112 In the United States, lineages with coli.114,116 Genomic studies of N. gonor- reduced susceptibility to cefixime were found to be rhoeae have identified isolates without known resis- primarily circulating among MSM, and transmis- tance determinants (Table 2), suggesting that in sion from MSM to men who have sex with women addition to GWAS, other methods, such as RNAseq (MSW) occurred more often than MSW to MSM and Tn-Seq, may be needed to identify the cellu- transmission.50 WGS of N. gonorrhoeae populations lar responses to and essential genes required under in New Zealand shows that the majority of clus- exposure to antibiotics.117–120 While the selective ters contain isolates from both men and women, pressures applied in the laboratory are unlikely suggesting the absence of clones associated with to fully replicate those pressures in the context of exclusively MSM transmission.23 While the N. gon- human infection and transmission, analysis of clin- orrhoeae global population does not show geo- ical N. gonorrhoeae isolates can identify loci under graphic structure, examining WGS of isolates from purifying or diversifying selection in the natural smaller scales (i.e., London) shows that transmis- environment. sion links are associated with shorter geographic Drug resistance alleles are likely to interact with distances.24 Beyond inferring likely transmission the genomic background and other resistance links, phylogenetic analyses utilizing a molecular determinants. For example, the presence of a clock enable additional inferences. Calculating the mosaic penA sequence is not a perfect predictor for time to most recent common ancestor (TMRCA) of reduced susceptibility to cefixime and ceftriaxone, isolates from pairs of known contacts in Sheffield, suggesting that additional variants contribute to UK enabled estimation of the average duration of this phenotype.22 In a model for MIC prediction, infection in this primarily heterosexual population some resistance alleles had synergistic interactions to be 3.4 months.107 where the presence of both alleles increased MIC WGS can also be used to track the spread of resis- more than the combination of their individual tance across geographic boundaries. For example, a effects, while other combinations did not increase cluster of cefixime resistant gonococcus mediated by MIC above levels associated with a single allele.103 mosaic penA XXXIV spreads from the west coast to For example, penB and mtrR promoter mutations the east coast of the United States.50 The TMRCA of appear to have a synergistic effect, and conversely, this cluster was estimated to be 1997, and the addi- the addition of rpsJ or mtrR mutationsdoesnot tion of subsequently sequenced isolates from the UK increase tetracycline MICs above levels of resistance confirmed this estimate.24 Nine percent of infections conferred by tetM alone. GWAS may be helpful to in Brighton, UK were found in transmission clusters identify additional variants that interact with resis- with U.S. isolates, indicating that intercontinental tance alleles, including compensatory and enabling

Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. 9 Genomics of multidrug-resistant Neisseria gonorrhoeae Mortimer & Grad

Table 2. Genomic studies of gonococcus

Resistant isolates without Location Year n known mechanisms Reference Accession

United States 2009−2010 236 – Grad et al.50 PRJEB2999 Canada 1982−2008 25 – Vidovic et al.159 – Canada 1989−2013 169 27 CRO-RS without Demczuk et al.160 PRJNA266539 mosaic penA Global 1982−2013 61 1 CFX-RS Ezewudo et al.21 SRA099559 1AZI-RS 1TET-R England 2014−2015 15 – Chisholm et al.78 – France 2010−2014 4 1 AZI-RS de Curraize et al.161 PRJEB13093 Canada 1989−2014 236 – Demczuk et al.162 SRP065041 UK 2004−2015 1842 – De Silva et al.24 PRJNA315363 UK 1995−2000 237 – Didelot et al.107 PRJEB2124 United States 2000−2013 1102 106 AZI-RS Grad et al.22 PRJEB7904 5 CIP-R Global 289 – Harrison et al.37 Previously published Europe 2009−2014 75 – Jacobsson et al.79 PRJNA322768 Global 14 – Unemo et al.139 PRJEB14020 UK 2014−2015 100 – Peters et al.111 Previously published United States 804 + 118 – Vidyaprakash et al.163 Previously and Brazil published Ireland 2008−2014 14 – Mac Aogain´ et al.164 PRJNA275092 New Zealand 2014−2015 398 2 CIP-R Lee et al.23 PRJNA394216 6Spec-RS 1 CFX-RS United States 2011−2016 61 – Papp et al.90 – (Hawaii) Australia 2005−2014 94 Kwong et al.165 PRJEB17738 Brazil 2006−2015 116 3 PEN-R Costa-Lourenc¸o et al.166 – Australia 2012−2014 94 – Buckley et al.112 PRJNA392203 UK 2014−2017 180 – Fifer et al.167 PRJEB23008 Australia 2011−2013 59 – Al Suwayyid et al.168 – Kenya 2010−2015 103 – Cehovin et al.169 PRJEB10104 Japan and 2015, 2017 4 – Lahra et al.170 PRJNA416507 Australia Note:Studieswereincludedwhen>1 isolates were sequenced. -R, resistant; -RS, reduced susceptibility.

mutations that allow resistant lineages to success- genetic loci and genome sequencing have further fully compete with their susceptible counterparts. supported mixed infections. Martin and Ison found WGS of patient samples may also illuminate the evidence of mixed infection using opa typing, which extent of within-host diversity and the impact of is based on restriction enzyme digestion of PCR mixed infections on pathogenicity, transmission, amplified opa genes.121,122 Different opa profiles and drug resistance. Horizontal gene transfer and were found in urethral and cervical swabs but not in recombination among N. gonorrhoeae provide evi- cultured samples. Using DNA–DNA hybridization dence of mixed infections, and several studies of of porB, Lynn and colleagues found evidence for

10 Ann. N.Y. Acad. Sci. xxxx (2018) 1–17 C 2018 New York Academy of Sciences. Mortimer & Grad Genomics of multidrug-resistant Neisseria gonorrhoeae mixed infection in 21% of samples.123 Using WGS, patients and public health authorities will need to De Silva et al. found evidence for distinct strains at be established. Rapid sharing of genomic data from different anatomical sites in 13% of pairs when mul- N. gonorrhoeae outbreakswillbekeytoenacttimely tiple samples were obtained from the same patient.24 interventions, but sequence data must be carefully The clinical importance of mixed infections remains deidentified and cleaned of human sequence data to unclear, though a concern about DNA-based resis- protect patient privacy. tance prediction is that these tests may have As a gonococcal vaccine would be a transfor- insufficient sensitivity to detect clinically meaning- mational intervention, much effort is being put ful resistant subpopulations. Within-host diversity into development of candidates. However, efforts may also interfere with reconstruction of transmis- to date have been stymied by N. gonorrhoeae’s sion networks when the genome of a single isolate evasion of the immune system through exten- per patient is sequenced.124 The amount of diversity sive variation in surface antigens. Two vaccines that accumulates during N. gonorrhoeae infection have proceeded to clinical trials in humans but and the reduction of diversity that occurs during were unsuccessful.126 Recently, a retrospective study transmission are unknown. The impact of diversity found that the outer membrane vesicle (OMV) on transmission inference is likely to be higher in meningococcal B vaccine showed some effectiveness networks containing asymptomatic patients where against infection by N. gonorrhoeae in New Zealand; duration of infection is longer. vaccination reduced the incidence of gonorrhea by Many public health laboratories are now incor- an estimated 31% compared to the unvaccinated porating WGS into pipelines to identify N. gonor- population.127 Reverse vaccinology, or the use of rhoeae and other pathogens. However, WGS of every microbial genomic information to identify vaccine patient sample may not be feasible in all settings, and targets,128 has been successfully used to develop other technologies for POC testing may prove to be vaccines for N. meningitidis serogroup B, a biva- faster and more economical for routine care. Further lent factor H binding protein (fHbp) vaccine and research is needed to determine the optimal role of a multicomponent vaccine.129–131 Two of the three WGS in public health, including establishing how main antigens from the multicomponent vaccine to sample the isolates for sequencing.58 For WGS to are also present in N. gonorrhoeae: fHbp and neisse- contribute to goals to reduce the burden of AMR N. rial heparin-binding antigen;132 however, some N. gonorrhoeae, results from WGS—including recon- gonorrhoeae isolates contain premature stop codons struction of transmission networks and inferences in these genes.133 The efficacy of OMV-containing about connections between demographic and geo- meningococcal B vaccines against N. gonorrhoeae graphic groups—must be reliable and translatable infection remains unknown but is of significant to cost-effective interventions. interest. Information gleaned from genomics could Further, routine WGS in clinical and public health be used to identify novel vaccine targets that are laboratories comes with a new set of technical and conserved across the N. gonorrhoeae population. ethical challenges. Introducing WGS into clinical Other efforts to develop vaccines, including from laboratories will require the development of user- nonprotein antigens, offer promising avenues of friendly tools to analyze sequence data and/or bioin- research.126 formatics training for clinical scientists; it will be Conclusion important to ensure that sequencing platforms and training on how to use them are widely available. With the rise of multidrug resistant N. gonorrhoeae, WGS directly from patient samples has similar eth- we urgently need to develop new tools for man- ical challenges to research, as agement of gonococcal infections—a need that sequences from both the patient as well as other extends from the development of new therapeutics pathogens may be captured.125 Additionally, dur- to improved diagnostics and public health inter- ing outbreak investigations, WGS can provide more ventions and strategies for how to implement these precise information than older molecular tech- tools optimally. WGS has the potential to contribute niques, and unreported transmission links may be to each of these efforts by improving our under- discovered.24 Guidelines for how to interpret and standing of N. gonorrhoeae at multiple scales, from report incidental findings from sequence data to the biology of the microbe to global evolution and

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