Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Hefei (2014–2015): genetic characteristics of antimicrobial resistance

Background Antimicrobial resistance (AMR) and genetic determinants of resistance of N. gonorrhoeae isolates from Hefei, China, were characterized adding a breadth of information to the molecular epidemiology of gonococcal resistance in China. Methods 126 N. gonorrhoeae isolates from a hospital clinic in Hefei, were collected between January, 2014, and November, 2015. The minimum inhibitory concentration (MIC) of N. gonorrhoeae isolates for seven antimicrobials were determined by the agar dilution method. Isolates were tested for mutations in penA and mtrR genes and 23S rRNA, and also genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Results All N. gonorrhoeae isolates were resistant to ciprofloxacin; 81.7% (103/126) to tetracycline and 73.8% (93/126) to penicillin. 39.7% (50/126) of isolates were penicillinase producing N. gonorrhoeae (PPNG), 31.7% (40/126) were tetracycline resistant N. gonorrhoeae (TRNG) and 28.6% (36/126) were resistant to azithromycin. While not fully resistant to extended spectrum cephalosporins (ESCs), a total of 14 isolates (11.1%) displayed decreased susceptibility to ceftriaxone (MIC ≥ 0.125 mg/L, n = 10), cefixime (MIC ≥ 0. 25 mg/L, n = 1) or to both ESCs (n = 3). penA mosaic alleles XXXV were found in all isolates that harbored decreased susceptibility to cefixime, except for one. Four mutations were found in mtrR genes and mutations A2143G and C2599T were identified in 23S rRNA. No isolates were resistant to spectinomycin. Gonococcal isolates were distributed into diverse NG-MAST sequence types (STs); 86 separate STs were identified. Conclusions N. gonorrhoeae isolates from Hefei during 2014–2015, displayed high levels of resistance to antimicrobials that had been recommended previously for treatment of gonorrhea, e.g., penicillin, tetracycline and ciprofloxacin. The prevalence of resistance to azithromycin was also high (28.6%). No isolates were found to be fully resistant to spectinomycin, ceftriaxone or cefixime; however, 11.1% isolates, overall, had decreased susceptibility to ESCs.


Background
Neisseria gonorrhoeae is the etiologic agent of gonorrhea, one of the most common bacterial sexually transmitted infections (STIs) worldwide. WHO estimated that 78.3 million of new cases of gonorrhea occurred among adults globally in 2012 [1].100, 245 cases of gonorrhea were reported nationally by the China Centers for Disease Control and Prevention in 2015, making it the fifth most commonly reported communicable disease in China [2]. At this time there are no effective vaccines for gonococcal infections and antimicrobial treatment continues to be the mainstay of control. However, N. gonorrhoeae has developed resistance to antimicrobials that had been used previously for treatment of this infection including sulfonamides, penicillins, tetracyclines and quinolones. Presently, extended spectrum cephalosporins (ESCs) that include ceftriaxone and cefixime are recommended as the first-line treatment of gonorrhea in most parts of the world. In many countries azithromycin has been added not just to treat chlamydia infection that often co-infects, but also to supplement treatment for N. gonorrhoeae itself [3]. Diminished susceptibility of N. gonorrhoeae and emergence of full resistance and treatment failures with ESCs (mainly pharyngeal gonorrhea) have been documented in several countries [4]. Furthermore, N. gonorrheae strains with high-level azithromycin resistance have been reported in France [5], the United States [6], Australia [7] and China [8]. Resistance to azithromycin threatens efficacy of dual antimicrobial therapy (ESCs plus azithrothmycin) that may result in decreased treatment options and enhance the possibility of untreatable infection.
A key component of a successful response plan to gonococcal antimicrobial resistance (AMR) is to conduct timely surveillance of resistance and treatment failures across geographic regions (e.g. GISP [U.S] and GASP (WHO/Europe) [9,10]) and to characterize the genetic elements of resistant strains. Antimicrobial susceptibility patterns vary by geographic region. AMR surveillance programs have been conducted in several cities in China [11,12]. The aims of the present study were to describe the prevalence of gonococcal AMR and the molecular epidemiological characteristics of N. gonorrhoeae strains from 2014 to 2015 in Hefei, a city in eastern China.

Clinical isolates
Clinical isolates of N. gonorrhoeae (n = 126) investigated in this study were collected consecutively from men with urethritis (urethral discharge and/or dysuria) and women with cervical infection in the STD clinic at Anhui Provincial Hospital, China, between January, 2014 and November 2015. Cotton swabs, used to obtain cervical and urethral specimens, were immediately streaked onto Thayer-Martin (T-M) selective medium to isolate N. gonorrhoeae. Inoculated plates were incubated at 36°C in 5% carbon dioxide for 24-48 h. and N. gonorrhoeae was identified by colonial morphology, Gram's stain, and oxidase testing. Gonococcal strains were subcultured onto chocolate agar plates, preserved in tryptone-based soy broth and then stored at −70°C until used.

Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MICs; mg/L) of N. gonorrhoeae isolates to penicillin, tetracycline, ciprofloxacin, spectinomycin, azithromycin, ceftriaxone and cefixime were determined on Difco GC medium base agar supplemented with 1% BBL™, IsoVitaleX™ enrichment (Becton, Dickinson and Company) using the agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) [13]. Gonococcal isolates were subcultured from frozen stocks onto chocolate agar and the resulting colonies were re-subcultured at 36°C in 5% CO 2 for 18-20 h before antimicrobial susceptibility testing was performed. Concentrations of antibiotics used were: penicillin, 0.06-8 mg/L; tetracycline, 0.125-16 mg/L; ciprofloxacin, 0.06-8 mg/L; spectinomycin, 4-128 mg/L; azithromycin,0.015-2048 mg/L; ceftriaxone, 0.002-0.5 mg/L and cefixime,0.002-0.5 mg/L. All antibiotics were purchased from Sigma Aldrich (USA), except for azithromycin, which was purchased from Shanghai yuanye Bio-Technology Co., Ltd. 95% ethanol was used to dissolve azithromycin powder to obtain a stock solution of 4096 mg/L; azithromycin stock was further diluted with distilled water to prepare twofold working dilutions. ATCC49226 and WHO reference strains G, K, M, O, P were used as quality controls for MIC determinations. Results were interpreted according to the CLSI standard, except for azithromycin that used breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); www.eucast.org [14].Criteria for decreased susceptibility to ceftriaxone (MIC ≥ 0.125 mg/L) and cefixime (MIC ≥ 0.25 mg/L) were defined by WHO [9].
Identification and typing of β-lactamase and tetM encoding plasmids β-lactamase production was determined by the paper acidometric method [15]. Isolates were classified as high level resistant to tetracycline (TRNG) if MICs were ≥16 mg/L and resistant (plasmid-mediated) to penicillin (PPNG) if they were β-lactamase positive [16]. The type of β-lactamase and tetM encoding plasmids were determined by PCR [17].
Genetic determinants associated with decreased susceptibility to ESCs and resistance of N. gonorrhoeaeto azithromycin Amplification of penA and mtrR genes and 23S rRNA were performed using published primers and conditions [18][19][20]. 14 gonococcal isolates with decreased susceptibility to ceftriaxone or cefixime were tested for penA mutations, including 3 isolates that had decreased susceptibility to both cefixime and ceftriaxone; all isolates were tested for mutations in mtrR and 41strains (including all 36 azithromycin-resistant and 5 randomly selected azithromycin sensitive strains) were tested for 23S rRNA mutations. PCR products were sequenced twice in both directions using an Applied Biosystems 3730XL DNA automatic sequencer. The nucleotide and deduced amino acid sequences were analyzed using the EditSeq program (LaserGene software [version 7.1; DNAStar Corp.]) and aligned against their respective prototypes using the Megalign program (LaserGene software). Standard sequences used for alignment in the study were: PBP2; wild strain LM306 (GenBank accession no. M32091 [18]); 4 alleles of 23S rRNA (GenBank accession no. AF450074 to AF450081 [20]) and mtrR (GenBank accession no. Z25796 [19]).

Molecular epidemiologic typing
N. gonorrhoeae multiantigen sequence typing (NG-MAST) was performed on all isolates (n = 126) as described [21]. The allele numbers of por and tbpB, and the sequence types (STs) were assigned using the NG-MAST website (www.ng-mast.net).

Statistical analysis
Statistical analysis was performed using statistical software IBM SPSS Statistics version 19.0 for comparisons of proportions. The level of significance was set at P < 0.05.
Characterization of resistance in penA and mtrR genes and 23S rRNA Mutations in the penA gene We sequenced the penA gene from 14 isolates that displayed decreased susceptibility to ceftriaxone (n = 10), cefixime (n = 1) or to both ESCs (n = 3) ( Table 2). Six PBP2 amino acid sequence patterns were identified, including a mosaic allele (XXXV) [18]. PBP2 allele XIII [19] was the predominant type (n = 5), followed by mosaic allele XXXV (n = 3) and alleles XVIII (n = 3), V (n = 1), XVII (n = 1) and XXI (n = 1) [19,22,23]. The three isolates that harbored mosaic allele XXXV had decreased susceptibility to cefixime; one (of the 3) displayed decreased susceptibility to both ceftriaxone and cefixime. Among 11 non-mosaic isolates that included 10 with decreased susceptibility to ceftriaxone and 1 to both ESCs, seven harbored alleles XIII, XVII and XXI and displayed an A501V mutation in PBP2. Three isolates with allele XVIII possessed an A501T mutation and one had a G542S mutation.

Mutations in domains of 23S rRNA
All 36 azithromycin-resistant isolates and five -susceptible isolates were tested for mutations in 23S rRNA. The A2143G (N. gonorrhoeae numbering) mutation was identified in four alleles in the13 isolates with high-level azithromycin resistance (MICs ≥ 256 mg/L). AC259 9 T mutation in four alleles was found in four isolates (MICs, 8 to 32 mg/L).No mutations were found in the five azithromycin-susceptible isolates.

Discussion
A high prevalence of resistance to antimicrobials previously used for the treatment of gonorrhea was documented in Hefei (2014-15) in this study: penicillin (68.9%); tetracycline (80.6%) and ciprofloxacin (100%). Similar levels of resistance to ciprofloxacin, tetracycline and penicillin have been reported in N. gonorrhoeae isolates from other cities in China: Nanjing (penicillin 67.7%, tetracycline 97.9% and ciprofloxacin 98.8%, in 2011-2012) [17]; Shanghai (penicillin 90%, tetracycline 82.56% and ciprofloxacin 100%, in 1988-2013) [11] and Guangzhou (penicillin 90.1% and ciprofloxacin 98%, in 2008-2013) [12]. A high percentage of PPNG and TRNG isolates was also identified in Hefei. Because penicillin and tetracycline are no longer used for the treatment of gonorrhea in China, continued high resistance may have been the result of having used these antimicrobials to treat other infections, particularly in patients who self-medicate, which is common in China [24].
Resistance to azithromycin was 28.6%, higher than had been reported from certain other countries, such as Japan [25] and Spain [26] during this period. We found that 10.3% (13/126) of isolates were highly resistant to azithromycin (MIC > 2048 mg/L). N. gonorrhoeae isolates with high-level azithromycin resistance have also been reported from other Chinese cities; Hanghzou [27] and Guangzhou [28]. Similar to other reports [29,30], our isolates with high-level azithromycin resistance all contained the mutation A2143G in 4 alleles in the peptidyltransferase loop of domain V of 23S rRNA. The C2599T mutation was found in 23S rRNA alleles in four moderately azithromycin-resistant N. gonorrhoeae isolates (representing MICs of 8, 16, and 32 mg/L).
There are no reports of ceftriaxone treatment failures in China. The percentage of isolates with decreased susceptibility to ceftriaxone (MIC ≥ 0.125 mg/L) was 10.3%   (13/126) in Hefei in 2014-2015, which is higher than the corresponding 4.5% reported from another eastern Chinese city, Nanjing, between 2011 and 2012 [17]. The proportions of ceftriaxone non-susceptible isolates (MIC ≥ 0.125 mg/L) in Shanghai, the largest city in eastern China, were 7%-13% from 2011 to 2013 [11]. This suggests the possibility that resistance to extended spectrum cephalosporins (ESCs) may be increasing in eastern China. PBP2 is targeted by cephalosporins and mutations in PBP2 may lead to a decline in susceptibility to ESCs [31]. Four (3.2%) of our isolates had reduced susceptibility to cefixime (MIC = 0.25 mg/L); three (ST12200, ST12647, ST13044) had the mosaic allele XXXV. The majority (7/11 [63.6%]) of non-mosaic isolates with decreased susceptibility to ceftriaxone harbored mutations in PBP2 at position 501 (A501V or A501T). Other studies have also found that A501 mutations in non-mosaic penA alleles play an important role in decreased sensitivity to ESCs [32]. A G542S mutation in PBP2 has been tentatively linked epidemiologically to higher ceftriaxone MICs [33] but an absolute correlation with a penA fixed-point mutation has not been shown. ST1407 N. gonorrhoeae strains, which display decreased susceptibility to ESCs or full resistance [34], and have spread worldwide, were not identified in Hefei. Five different STs (ST10367, ST12649, ST12650, ST12657, ST13132) that shared the same tbpB110 with ST1407 were identified but their por alleles were different (by 13 to 37 nucleotides) than por908 of ST1407; all isolates with these STs were susceptible to both ceftriaxone and cefixime. ST1407, however, has been isolated in Shanghai [35]. Significant diversity of isolates was identified among 126 isolates that were divided into 86 NG-MAST STs. Notably, ST1866 has been reported only from China and identified to be associated with high level azithromycin resistance [28]. The 5 ST1866 isolates in this study were azithromycin-resistant strains; three had high level resistance to azithromycin.

Conclusions
This is the first study that describes antimicrobial resistance profiles and molecular determinants of N. gonorrhoeae strains isolated from Hefei, China. N. gonorrhoeae isolates from Hefei, during 2014-2015, possessed high level resistance to antimicrobials previously recommended for the treatment of gonorrhea, e.g., penicillin, tetracycline and ciprofloxacin. Azithromycin-resistance was documented in 28.6% of isolates, higher than that reported from other areas of the world. No isolates were found to be resistant to ceftriaxone, cefixime or spectinomycin. However, 11.1% isolates had decreased susceptibility to ESCs, which was often associated with mutations in the penA gene. Gonococcal isolates were distributed into diverse NG-MAST sequence types.