First description of a cefixime- and ciprofloxacin-resistant Neisseria gonorrhoeae isolate with mutations in key antimicrobial susceptibility–determining genes from the country of Georgia

Antimicrobial resistance in Neisseria gonorrhoeae is a global health problem. Enhanced international collaborative surveillance and disease control are needed to reduce the global burden of this important pathogen. Currently the antimicrobial resistance properties and molecular mechanisms of multidrug-resistant N. gonorrhoeae in the Republic of Georgia represent a significant knowledge gap. Here we report the isolation of a strain of N. gonorrhoeae exhibiting resistance to cefixime and ciprofloxacin with reduced susceptibility to penicillin and tetracycline from a patient being treated at a Georgian medical centre. Notably, this isolate was found to contain a mosaic penA allele and to harbour mutations in genes conferring susceptibility to the β-lactam, cephalosporin, fluoroquinolone, macrolide and penicillin classes of antibiotic. To our knowledge, this is the first report to describe the key mutations conferring the antimicrobial resistance properties of an isolate of N. gonorrhoeae from Georgia.


Introduction
Historically, Neisseria gonorrhoeae has been associated with a high rate of morbidity in military populations [1,2]. Soldiers with urogenital infection can enter into a state of debilitation preventing them from performing their duties [3]. Importantly, untreated or incompletely treated N. gonorrhoeae infection can lead to significant and potentially life-threatening sequelae including pelvic inflammatory disease, epididymitis and infertility [4]. Disseminated gonococcal infection can lead to septic arthritis and in rare cases septic shock and death [5]. Accurate data regarding the prevalence of sexually transmitted infections in the Georgian military population is currently lacking. The acquisition of such data is essential for the development and deployment of rational intervention strategies [6]. To this end, a collaborative study was initiated between the Georgian Ministry of Defense military hospital in Gori, the US Army Medical Research Directorate in Georgia (USAMRD-G) and the Uniformed Services University of the Health Sciences in Bethesda, Maryland, with the goal of characterizing N. gonorrhoeae isolates derived from men being treated for urethritis at the Gori facility. During this study, an unusual isolate of N. gonorrhoeae was recovered from a urethral swab taken from a 23-year-old soldier. This isolate displayed resistance to cefixime and ciprofloxacin and reduced susceptibility to penicillin and tetracycline. In order to further characterize this isolate, a detailed molecular and phenotypic analysis was performed. The N. gonorrhoeae multiantigen sequence type (NG-MAST) was determined, and the nucleotide sequence of genes associated with vulnerability to the cephalosporins and the fluoroquinolones were determined [7,8].
Furthermore, the presence of mutations conferring reduced susceptibility to these antimicrobials was uncovered [9][10][11][12][13][14]. To our knowledge, this is the first study to describe an N. gonorrhoeae isolate containing previously described fluoroquinolone and cefixime resistance mutations from the Republic of Georgia.

Inclusion criteria
All male active-duty service members older than 18 years who presented to the military hospital with the symptoms of acute urethritis were offered enrollment. Acute urethritis was defined as having one or more of the following symptoms: urethral discharge, dysuria, penile irritation or watery, viscous penile secretions.

Exclusion criteria
Male subjects younger than 18 years of age and female subjects were excluded from the study.

Laboratory testing
First-void urine and urethral swabs were collected from all patients. Urethral swab specimens were Gram stained and cultured on modified Thayer Martin medium at the military hospital. Cultures were transferred to the USAMRD-G laboratory in Tbilisi for identification and antimicrobial susceptibility testing. Nucleotide sequence analysis of the gyrA, parC, ponA, penB, mtr and penA alleles was performed at the Uniformed Services University of the Health Sciences in Bethesda, Maryland, using previously published primers [15][16][17][18][19][20][21], as was NG-MAST, which was also performed according to previously published guidelines [20,22]. Susceptibility testing was performed using Etest (bioMérieux, Hazelwood, MO, USA), and confirmation of the cefixime MIC was done by agar dilution and interpreted using the 2017 guidelines of the Clinical Laboratory Standards Institute [23,24]. Alignments of predicted amino acid sequences of penA alleles was performed using the CLUSTAL 0 (1.2.4) multiple sequence alignment tool [25,26].

Case report
A 23-year-old male soldier reported to the military hospital in Gori presenting with symptoms consistent with urethritis. He indicated that he had an acute urethral discharge and that he was experiencing burning sensations during urination. The patient indicated that he had been treated for Chlamydia in the past and that he had unprotected sexual contact with a female sex worker at least 10 days before symptom onset. The patient was offered enrollment in the USAMRD-G study and consented. A urethral swab and first-void urine were collected, and he was empirically treated with a combination of ceftriaxone and levofloxacin. The patient was discharged from the hospital and subsequently lost to follow-up. The urethral swab and urine samples were transferred to the Gori hospital laboratory for analysis and culture. Isolates consistent with Neisseria sp. were obtained from the urethral swab and forwarded to USAMRD-G for confirmation and characterization.

Results
Gram stain results from the initial urethral swab indicated the presence of Gram-negative diplococci, and biochemical testing of the resulting isolates revealed that they were both catalase and oxidase positive. These results were found to be consistent with members of the family Neisseriaceae, and further analysis was undertaken to confirm these results and to identify the species [17]. Targeted biochemical analysis using a Neisseria-and Haemophilus-specific panel (API NH; bio-Mérieux) designed to interrogate bacterial metabolism indicated that the genus and species specific reactions of the isolate were consistent with N. gonorrhoeae [18]. Susceptibility testing by Etest revealed that the isolate was resistant to ciprofloxacin and displayed intermediate-level resistance to cefixime, benzylpenicillin and tetracycline ( Table 1). The cefixime MIC was also determined by agar dilution and was found to be at a resistant level (MIC 0.5 μg/mL) ( Table 1). Molecular typing based on NG-MAST, which targets the porB and tbpB genes, were consistent with NG-MAST sequence type (ST) 2212 [20,22]. Intriguingly, gonococcal isolates of ST2212 with reduced susceptibility to the fluoroquinolone class of antibiotics were previously reported in Estonia [19]. The finding of a similar isolate in Georgia may be indicative of a general trend towards reduced fluoroquinolone susceptibility in Eurasian Neisseria isolates. Indeed, sequencing of the quinolone resistance-determining region identified three substitutions that are associated with reduced susceptibility to fluoroquinolones (gyrA substitutions S91F/D95G and parC substitution S87R) ( Table 2) [9]. Sequencing of four genes associated with increased resistance to penicillin, ponA, penB, mtr and penA, was also performed. Analysis of the ponA (penicillin-binding protein 1) sequence revealed a leucine to proline substitution (L421P) previously associated with reduced penicillin susceptibility [10]. Glycine to lysine (G210K) and alanine to histidine mutations (A121N) were detected in penB (porB1b). These mutations have previously been found to reduce susceptibility to β-lactam and tetracycline antibiotics when they occur in the presence of mutations in ponA and the mtr locus [27,28]. Importantly, a single base pair deletion (−T/A) was identified in the −35 region of mtrR. This deletion is known to decrease expression of the repressor protein MtrR, resulting in increased expression of the MtrCDE efflux pump, leading to decreased susceptibility to macrolides and β-lactams [27]. Furthermore, a mosaic penA (penicillin-binding protein 2) allele was also identified [12,29,30]. The penA allele of this isolate encodes 53 amino acid substitutions and is most closely related to the mosaic penA allele XXXIV (Fig. 1) [8].

Discussion
To our knowledge, this is the first published report of a multidrug-resistant gonococcal isolate identified in the Republic of Georgia. This isolate, named G093, carries a mosaic penA allele and other key mutations linked to antibiotic resistance that have not been previously reported in Georgia, including the S91F and D95G mutations in gyrA and a S87R mutation in parC. The penA XXXIV allele is associated with reduced susceptibility to extended-spectrum cephalosporins, but not ceftriaxone resistance [8]. F89 is a ceftriaxoneresistant strain isolated in France that encodes the penA allele CI, which is nearly identical to penA XXXIV but includes an A501P substitution resulting from a point mutation at nucleotide 1501 [12,29]. Because residue 501 has been shown to be critical for ceftriaxone resistance, it is possible that the acquisition of one additional point mutation by strain G093 at nucleotide 1501 would yield a novel ceftriaxone-resistant Georgian isolate. The undetected development of such isolates among commercial sex workers would be a significant event and could possibly initiate the end of the utility of the expanded-spectrum cephalosporins as treatment options for gonorrhoea in Georgia.
The results presented in this study are of regional significance given that little is known about the aetiology of sexually transmitted infections and the mechanisms of antibiotic resistance in Georgia. Our results are of broad significance given that the infection was most likely acquired from a sex worker, as sex workers often engage in sexual activities with multiple partners, increasing the chance that organisms with these mutations have spread throughout the country and beyond. Therefore, the identification of the isolate reported here may indicate the potential emergence and spread of a fluoroquinolone-and cephalosporin-resistant N. gonorrhoeae strain in Georgia. This report underscores the need for identification and susceptibility testing to be conducted as part of the treatment algorithm for suspected N. gonorrhoeae infections. Empirical treatment without susceptibility testing can lead to the emergence of strains with reduced susceptibility and decrease the efficacy of currently available antimicrobial agents [12,30,31]. Further research will be required to determine the degree of antimicrobial resistance in the Georgian N. gonorrhoeae population, the geographic spread of antimicrobial resistance and the most common resistance mechanisms. It is recommended that identification and susceptibility testing be conducted on all isolates obtained from patients experiencing acute urethritis, particularly when gonorrhoea is suspected. Reduced susceptibility to β-lactams and tetracyclines penA Penicillin-binding protein 2 53 independent amino acid substitutions Reduced susceptibility to extended-spectrum cephalosporins