Gonorrhea is the second most prevalent bacterial sexually transmitted infection (STI) worldwide, with an estimated 87 million infections in 2016 [1
]. In the United States, 555,608 cases of gonorrhea were reported to the Centers for Disease Control and Prevention (CDC) in 2017, a 67% increase from 2013 [2
]. Neisseria gonorrhoeae
(NG) has progressively developed resistance to all commonly-prescribed antimicrobials [3
] and is considered as one of the top three urgent threats among antibiotic-resistant bacteria [4
In response to the threat of multidrug-resistant NG, the World Health Organization (WHO) has proposed a global action plan, comprising several strategies, to mitigate the emergence and spread of antimicrobial resistant (AMR) NG [5
]. One of the strategies proposed by the WHO to combat AMR NG is the development of molecular methodologies for monitoring and detecting antimicrobial resistance in NG. Traditionally, the determination of AMR NG has been performed by minimum inhibitory concentration (MIC) testing, which requires viable organisms for proper execution. A well developed and validated molecular method for AMR determination could obviate the requirement for a viable clinical isolate. It has been hypothesized that antimicrobial susceptibility testing (AST) at the point-of-care (POC) could lead to precision treatment, i.e., utilizing specific antibiotics based on the AMR profile of NG as opposed to syndromic management of NG infections, including reusing previously recommended antimicrobials. This approach could reduce ceftriaxone selection pressure as ceftriaxone is one of the few remaining antibiotics effective against NG, and help delay the emergence of extended spectrum cephalosporin (ESC) resistance or untreatable gonorrhea [6
Ciprofloxacin, a previously recommended antimicrobial, is an excellent candidate for precision treatment as 69.9% of NG isolates collected by the gonococcal isolate surveillance project (GISP) in 2017 were susceptible to ciprofloxacin [8
]. Furthermore, ciprofloxacin susceptibility can be reliably predicted through the detection of genetic markers, thus allowing for the characterization of ciprofloxacin susceptibility directly from a variety of clinical specimens, including those used for the nucleic acid amplification test (NAAT)-based detection of gonorrhea [9
]. At the molecular level, resistance to ciprofloxacin is strongly associated with a single mutation at codon 91 of the gyrase A
) gene [10
], and detection of this mutation has been shown to be an excellent predictor of ciprofloxacin resistance [9
]. Although other codons in gyrA
, as well as in topoisomerase IV (parC
), and in rare instances, penicillin binding protein 2 (PBP2/PenA
) have been associated with resistance to ciprofloxacin, the vast majority of ciprofloxacin-resistant NG harbor the single mutation at codon 91 of GyrA
, making this gene the target of choice for molecular screening [3
]. As such, in 2016, a health care-based system study showed that implementation of a single molecular assay testing for gyrA
mutations could be utilized in clinical care, and reduced the use of ceftriaxone while increasing the use of ciprofloxacin [11
]. Furthermore, patients with wildtype gyrA
NG who were treated with ciprofloxacin successfully cleared the infection at all anatomical sites of infections [12
]. Despite the successful use of ciprofloxacin in clinical settings, this approach has not been evaluated in STI clinic settings, where this approach could potentially be highly effective.
Baltimore, Maryland, a large city with a high prevalence of gonorrhea (691.7/100,000 in 2017) [13
], could be an excellent setting for the re-use of ciprofloxacin as a treatment option. According to our previous study, over 55% of the NG isolates collected in 2016 were susceptible to ciprofloxacin [14
]. However, additional studies are necessary to better define the epidemiology of ciprofloxacin-resistant NG in Baltimore and determine whether ciprofloxacin could be effectively re-used in this particular population. In this study, we report the epidemiology of ciprofloxacin resistance in Baltimore through the molecular analysis of 510 NG isolates collected between 2014 and 2016. Additionally, clinical characteristics and demographics were evaluated to identify correlates of ciprofloxacin-resistant NG infections.
A total of 510 urethral NG isolates collected from 2014 to 2016 were included in the study. The isolates were recovered from 510 male subjects (15–69 years old), predominately African American (96.5%), heterosexual (85.7%), and HIV-uninfected (92.5%) (Table 1
). The majority of participants (96.7%) reported symptoms of urethritis at the time of sample of collection, and 4.3% were co-infected with syphilis.
The number of isolates collected by year was uniformly distributed: 170 (33.3%), 185 (36.3%), and 155 (30.4%), collected in 2014, 2015, and 2016, respectively. Genotypic typing by PCR revealed that 32.4% (165/510) of the isolates had mutation(s) in the gyrA
gene. The percentage of isolates with gyrA
mutant sequences was 24.7% (42/170), 28.7% (53/185), and 45.2% (70/155) in 2014, 2015, and 2016, respectively. The increase in the percentage of gyrA
mutant NG from 2014 to 2016 was statistically significant (p < 0.001) in a bivariate and multivariate regression analysis (Table 2
and Table 3
). Multivariate analysis was specifically used to determine the predictor of our outcomes adjusted for co-variates period of the 165 NG isolates with mutant gyrA
sequences; 63.6% (105/165) were viable for susceptibility testing, and 98.1% (103/105) were confirmed as ciprofloxacin resistant by the E-test method. The remaining two isolates displayed intermediate resistance to ciprofloxacin. Susceptibility testing of 92 randomly selected isolates with gyrA
wildtype sequences showed that all isolates were susceptible to ciprofloxacin.
Stratification of the isolates by gyrA
genotype revealed that older age was associated with a mutant gyrA
genotype (p = 0.002), suggestive of an association between ciprofloxacin resistance and age (Table 2
), and participants ≥35 years of age were 2.35 times (95% CI, 1.47–3.76) more likely to have a gyrA
mutant NG infection than younger participants (p < 0.001) (Table 3
). Race, sexual orientation, symptomology, or co-infection with another STI (HIV or syphilis) were not associated with a particular NG gyrA
genotype (Table 2
As the first step in the development of a POC test for gonorrhea diagnosis and antimicrobial susceptibility testing, we sought to determine whether ciprofloxacin could be a suitable antimicrobial for precision treatment in Baltimore. Using a molecular approach, we have shown low to moderate levels of ciprofloxacin resistance in Baltimore during a three-year period, suggesting that this antimicrobial might be a suitable option for precision treatment. Furthermore, our study identified an association between age and ciprofloxacin-resistant NG infections.
The overall percentage (32.4%) of mutant gyrA
NG infections, and thus resistance to ciprofloxacin, in our study is similar to the nationwide percentage (30.1%) reported by GISP in 2017 [8
]. The high percentage of wildtype gyrA
(ciprofloxacin-susceptible) NG isolates in this study provides further evidence for the use of ciprofloxacin for targeted precision treatment, which may help to delay the emergence and spread of resistance to current first-line regimens [6
]. Additionally, use of ciprofloxacin could make partner therapy easier. However, the increase in the percentage of isolates with mutated gyrA
sequences from 2014 to 2016 (24.7 to 45.2%), which is consistent with national trends [15
], suggests that ciprofloxacin resistance has persisted despite ciprofloxacin not being used as a recommended treatment option for gonorrhea. Therefore, the reintroduction of ciprofloxacin as a gonorrhea treatment option will require enhanced surveillance practices to determine if and when the targeted treatment is no longer a viable option.
Our study also identified age as an important demographic correlate associated with ciprofloxacin-resistant NG infections. Men older than 35 years of age were 2.35 times more likely to have a mutant gyrA
NG infection than younger men. On the contrary, men under 24 years of age were more likely to have a wildtype gyrA
(ciprofloxacin-susceptible) infection. These findings suggest that younger individuals (<24 years of age) may be the ideal population for targeted precision treatment with ciprofloxacin. A targeted treatment approach may prove efficacious in older individuals, but caution is warranted considering the higher percentage of ciprofloxacin resistance observed in this study and the results of previous studies which have reported an association between older age and antimicrobial-resistant NG infections [16
]. Contrary to previous studies [16
], our study did not find an association between ciprofloxacin-resistant NG and sexual orientation; however, the number of NG isolates from men who have sex with men (MSM) was limited, and a larger study focusing on MSM may provide more details.
The re-introduction of ciprofloxacin as a treatment option could help to mitigate the emergence and spread of AMR NG. According to a modeling study, the introduction of a POC ciprofloxacin susceptibility test could help to decrease the use of ceftriaxone by as much as 66%, thus potentially helping to extend the usefulness of this antimicrobial [17
]. However, caution is warranted since the introduction of a POC test targeting a single antimicrobial, such as ciprofloxacin, may accelerate the emergence of triply-resistant (ciprofloxacin, ceftriaxone, and azithromycin) NG isolates [7
]. These modelling studies highlight the need for the development of POC tests for resistance to multiple antimicrobials, but the complex resistance mechanisms of other antimicrobials, such as ceftriaxone [3
], have hindered the development of such molecular tests. Phenotypic tests, on the other hand, may determine resistance to multiple antimicrobials, but are not currently available at the POC. Therefore, until POC tests targeting multiple antimicrobials can be developed, a single antimicrobial POC test, capable of providing an alternative treatment option, such as ciprofloxacin, might be the most suitable option. The development of a POC test for gonorrhea and identification of ciprofloxacin susceptibility is attractive because ciprofloxacin can be administered before the patient leaves the clinic.
Our study had several limitations. First, all of the isolates were collected in Baltimore, Maryland at one clinic, thus limiting the generalizability and scope of these results. However, the data reported by GISP in 2017 suggest low levels of ciprofloxacin resistance in the US [8
]. Second, we had limited access to epidemiological, complete demographic, and behavioral data, which limited the scope of our analysis. It should also be noted that these samples were collected between 2014 and 2016; it is likely that the levels of ciprofloxacin susceptibility and resistance have varied since then, and these results should be viewed with that caveat. Finally, samples from women and samples from extragenital sites (pharyngeal and rectal) were not available for this analysis. Additionally, a more complete approach to the study would have been to expand molecular testing to include ParC
targets associated with ciprofloxacin resistance, as well as the rare PenA
target associated with ciprofloxacin resistance. However, as stated previously, the vast majority of ciprofloxacin-resistant NG harbor the single mutation at codon 91 of GyrA
, and focusing on this target, as similar studies have shown, proves to be just as effective at identifying ciprofloxacin susceptibility and resistance, as expanded tests show.