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Antimicrobial Susceptibility Evaluation and Multiple-Locus Variable Number Tandem Repeat Analysis of Neisseria gonorrhoeae Isolates in China in 2012

Yu, Rui-xing MD; Yin, Yueping PhD; Dai, Xiu-qin; Chen, Shao-chun PhD; Han, Yan PhD; Zheng, Bing-jie PhD; Zhang, Guo-yi MD; Chen, Xiang-sheng PhD

Sexually Transmitted Diseases: April 2017 - Volume 44 - Issue 4 - p 197–201
doi: 10.1097/OLQ.0000000000000580
Original Study
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Objective This study aimed to gain information on the antimicrobial susceptibility and molecular epidemiological typing of Neisseria gonorrhoeae (NG) isolates in China in 2012.

Methods A total of 244 NG isolates were consecutively recovered from patients with uncomplicated gonorrhea attending sexually transmitted disease clinics in 3 Chinese cities—Guangzhou, Nanjing, and Tianjin—in 2012. Neisseria gonorrhoeae susceptibilities to penicillin and tetracycline were examined by detecting penicillinase-producing NG (PPNG) and high-level tetracycline-resistant NG, and NG susceptibilities to ciprofloxacin, spectinomycin, ceftriaxone, and cefixime were determined using an agar dilution method. Neisseria gonorrhoeae isolates were typed by multiple-locus variable number tandem repeat analysis. We conducted a χ2 analysis to compare clusters with Bonferroni correction and Kruskal-Wallis test.

Results Neisseria gonorrhoeae isolates gathered from the 3 cities differed significantly in the prevalence of tetracycline-resistant NG (P < 0.001) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.001). The analysis of the combination of the 7 variable number of tandem repeats loci for all of the 244 isolates yielded 110 multiple-locus variable number tandem repeat analysis types falling into 5 clusters. Cluster III was associated with PPNG, whereas cluster II was associated with non-PPNG (P < 0.05) and NG treated with ceftriaxone with a minimum inhibitory concentration of 0.125 mg/L or higher (P < 0.05).

Conclusions Antimicrobials that can be used with confidence to treat NG infection currently in China include ceftriaxone and spectinomycin, but not penicillin, tetracycline, ciprofloxacin, and cefixime. Moreover, some of the resulting clusters were associated with PPNG and NG with decreased ceftriaxone susceptibility.

We conducted a study of antimicrobial susceptibility evaluation and multiple-locus variable number tandem repeat analysis of 244 Neisseria gonorrhoeae isolates collected from Tianjin, Nanjing, and Guangzhou, China, in 2012.

From the *Department of Dermatology, China-Japan Friendship Hospital, Beijing; and †National Center for STD Control, Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Dermatology, Nanjing, China

Conflict of interest: None declared.

Correspondence: Yueping Yin, PhD, Reference Laboratory, National Center for STD Control, Chinese Academy of Medical Sciences and Peking Union Medical College Institute of Dermatology, 12 Jiangwangmiao St, Nanjing 210042, China. E-mail: yinyp@ncstdlc.org.

Received for publication November 19, 2014, and accepted December 5, 2016.

Neisseria gonorrhoeae (NG) is causative of gonorrhea. There were more than 90,000 new NG infections in China in 2014, whose prevalence in China stands second among all sexually transmitted diseases (STDs).1 The recommended antibiotics and doses for treating NG infection are as follows: 1-g ceftriaxone in Japan, 250-mg ceftriaxone combined with 1-g azithromycin in the United States, 500-mg ceftriaxone combined with 2-g azithromycin in Australia as well as in European countries, 250-mg ceftriaxone coadministered with 1-g azithromycin in Canada, and 250-mg ceftriaxone in China.2–6 Although effective antimicrobials are the most important measures to prevent STD from spreading, the emergence of antimicrobial-resistant NG strains7,8 necessitates continuous monitoring of their susceptibilities to antibiotics, including spectinomycin, cefixime, and ceftriaxone, which were previously used and/or are currently used to treat this infection worldwide.

There were multiple typing methods including multiple-locus variable number tandem repeat analysis (MLVA).9 Dependent on determination of variable number of tandem repeats (VNTR) at each locus in the total genome of bacteria,10–14 MLVA is a rapid, inexpensive typing method and has been used to genotype many bacterial species, including Neisseria meningitidis10–14 and NG.15–20

Aiming at gaining new information on the molecular epidemiological typing and antimicrobial susceptibility of NG isolates in China, in this study, we collected 244 NG isolates from 3 Chinese cities; investigated their susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, and ceftriaxone; identified variables associated with NG with decreased ceftriaxone susceptibility; and typed isolates by means of MLVA.

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METHODS

Bacterial Strains

In 2012, a total of 244 NG isolates were consecutively recovered from patients with uncomplicated gonorrhea attending STD clinics of Guangzhou Dermatology Hospital, Chinese Academy of Medical Sciences Institute of Dermatology, and Tianjin Centers for Disease Control and Prevention (CDC) in the cities of Guangzhou, Nanjing, and Tianjin, respectively, which lie far apart in China—Guangzhou, adjacent to Hong Kong, is the capital of the southern province of Guangdong; Nanjing, close to Shanghai, is the capital of the eastern province of Jiangsu; and Tianjin, near Beijing, is a northern municipality. This collection of isolates consisted of 71 from Guangzhou, 103 from Nanjing, and 70 from Tianjin, of which 92.2% were isolated from the urethra of male patients and the rest from the cervix of female patients. In addition, 1 NG isolate was collected from 1 patient. The mean age of male patients was 36.3 years (range, 19–60 years), and the mean age of female patients was 32.5 years (range, 20–55 years). Before the study was first conducted, ethics permission was obtained from the research ethics committee of the Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College. We informed the patients about this study at every participating site with written notices. The collect, identify, confirm, and store methods for NG isolates were as previously described.21

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Antimicrobial Susceptibility Testing

The β-lactamase—a bacterial enzyme conferring resistance to β-lactam penicillin—produced by NG isolates was detected using a paper acidimetric testing method.21 Antimicrobial susceptibility testing was performed for tetracycline, ciprofloxacin, spectinomycin, and ceftriaxone using an agar dilution method according to the guidelines of World Health Organization (WHO) West Pacific Region Resistance Surveillance Programme.22Neisseria gonorrhoeae susceptibility to cefixime was tested according to the Clinical and Laboratory Standards Institute guidelines.23 Each testing involved the following control strains: the international NG reference strain ATCC49266 and WHO reference strains G (tetracycline-resistant NG [TRNG]), J (penicillinase-producing NG [PPNG]), L (strain with decreased ceftriaxone susceptibility and high-level resistance to ciprofloxacin), and K (strain nonsusceptible to cefixime).24Neisseria gonorrhoeae antimicrobial susceptibility was determined according to the criteria set by the WHO West Pacific Region Resistance Surveillance Programme guidelines. Isolates treated with ciprofloxacin with a minimum inhibitory concentration (MIC) of 1.0 mg/L or higher or spectinomycin with an MIC of 128 mg/L or higher were considered resistant; those treated with ciprofloxacin with an MIC of 0.06 to 0.50 mg/L were classified as isolates with decreased susceptibility; and those treated with tetracycline with an MIC of 16 mg/L or higher were classified as TRNG, although those treated with tetracycline with an MIC of 2 mg/L or higher were also considered resistant. The strains were defined as NG susceptible and nonsusceptible to ceftriaxone and cefixime as described elsewhere.23

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Multiple-locus Variable Number Tandem Repeat Analysis

The MLVA was performed using previously reported VNTR loci: VNTR264, VNTR471, VNTR947, VNTR1609, VNTR1638, VNTR2048, and VNTR2168. The primers used to amplify each of the 7 loci were the same as those described in a previous study.15 Each polymerase chain reaction (PCR) was run in a final reaction volume of 20 μL with a “dyad” thermocycler, and electrophoresis was performed as reported in the previous study.15 The gels were stained using a silver staining method.25 The DNA fragments required for performing MLVA on all isolates were successfully amplified by PCR, and the lengths of PCR products were converted into numbers of repeats. A 7-digit profile was obtained for each isolate, indicating that the applied MLVA scheme had 100% typeability.

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Data Analysis

The χ2 tests were conducted to compare differences in the prevalence of antimicrobial resistance for selected antibiotics. Cluster analysis, including construction of a dendrogram, was performed using unweighted pair group method with arithmetic mean. Associations between clusters and susceptibility levels were examined with χ2 tests. To investigate associations between clusters and epidemiological factors, χ2 tests were conducted for testing independence, and Kruskal-Wallis tests were performed for continuous variables. Fisher exact tests were conducted when expected values were less than 1. Bonferroni correction was used to adjust for multiple comparisons. All descriptive and statistical analyses were performed using SPSS 20.0 (IBM, Chicago, Ill), and graphs were created using Excel 2013 (Microsoft, New York, NY) and the STATISTICA software.

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RESULTS

Antimicrobial Susceptibility of NG Isolates

No statistically significant differences in the prevalence of PPNG were noted among isolates from Guangzhou, Nanjing, and Tianjin (χ2 = 1.714, P = 0.424) (Table 1). The prevalence of TRNG significantly differed by sampling location (χ2 = 26.418, P < 0.001), which was highest (44.3%) in Guangzhou, closely followed by Nanjing (40.8%), and lowest in Tianjin (8.5%). The prevalence of ciprofloxacin-resistant NG was 98.6% in Guangzhou and 100% in both Nanjing and Tianjin. The prevalence of NG susceptible to spectinomycin was 100% in each sampling city. Tands-onhe prevalence of NG susceptible to ceftriaxone with an MIC of 0.125 mg/L or higher was highest in Guangzhou (28.6%), intermediate in Nanjing (15.5%), and lowest in Tianjin (2.8%) (χ2 = 17.782, P < 0.001). The prevalence of NG susceptible to ceftriaxone was 97.2% (69/71) in Guangzhou and 100% in Nanjing and Tianjin. The prevalence of NG nonsusceptible to cefixime was not significantly different in the 3 cities (χ2 = 1.546, P = 0.462), and the mean prevalence was 25.4%.

TABLE 1

TABLE 1

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Multiple-locus Variable Number Tandem Repeat Analysis Types

The MLVA was performed on all of the 244 NG isolates at 7 VNTR loci. The number of repeats at different VNTR loci ranged from 2 (VNTR264, VNTR1609, VNTR1638, and VNTR2148) to 4 (VNTR471 and VNTR947). The analysis of the combination of the 7 VNTR loci for all isolates resulted in 110 MLVA types, which varied in the number of repeats of at least 1 locus. Fifty-nine MLVA types represented only 1 isolate each, and the rest contained 2 to 16 isolates each. Multiple-locus variable number tandem repeat analysis type 25 (1, 3, 2, 1, 2, 2, 2) was the most prevalent, shared by 16 isolates from the 3 cities—5 from Guangzhou, 9 from Nanjing, and 2 from Tianjin.

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Multiple-locus Variable Number Tandem Repeat Analysis Data and NG Antimicrobial Resistance

A minimum spanning tree was created according to MLVA profiles, with each of the 7 VNTR loci assigned an equal weight (Fig. 1). Based on Euclidean distance 2.0, 5 clusters (I–V) were yielded. As shown in Table 2, these clusters included 98.8% of the total 244 isolates, with 63, 41, 85, 39, and 13 isolates falling into clusters I, II, III, IV, and V, respectively, whereas 3 isolates were excluded from them. Cluster III was associated with PPNG (χ2 = 9.827, P = 0.002), whereas cluster II was associated with non-PPNG (χ2 = 5.029, P = 0.0025) and with NG treated with ceftriaxone with an MIC of 0.125 mg/L or higher (χ2 = 7.362, P = 0.007).

Figure 1

Figure 1

TABLE 2

TABLE 2

The 5 clusters were not statistically significantly different in the prevalence of TRNG and NG susceptible to cefixime. In terms of the distribution of ceftriaxone MICs in isolates in these clusters, the MIC peaked at 0.032 mg/L in isolates in clusters I (35.00%) and V (46.20%), but at 0.064 mg/L in clusters II (39.00%), III (42.4%), and IV (35.9%).

The 5 clusters significantly differed in the proportion of isolates from males (P = 0.035), ranging from 84.1% in cluster I to 100% in cluster II, and in the rate of patients with antibiotic use 1 month ago (P = 0.022), varying from 5.1% in cluster IV to 29.3% in cluster II. The prevalence of TRNG and NG susceptible to cefixime in the 5 clusters was not statistically significantly different across the 5 clusters when stratified by age, marital status, and clinical course (Table 3).

TABLE 3

TABLE 3

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DISCUSSION

Efficacious antibiotics are fundamental to successful treatment of NG infection. In the present study, STD clinic attendees in Guangzhou, Nanjing, and Tianjin had high prevalence of PPNG and TRNG and 100% prevalence of ciprofloxacin-resistant NG in 2012. This suggests that penicillin, tetracycline, and ciprofloxacin should no longer be used to treat NG infection in these areas. On the other hand, none of the isolates gathered from the 3 cities developed resistance to spectinomycin, but 2 NG isolates were resistant to ceftriaxone, implicating that the 2 antimicrobials can be used as first-line treatment for NG infection there. However, the mean prevalence of NG treated with ceftriaxone with an MIC of 0.125 mg/L or higher in these cities reached a high level of 15.6%, which could lead to the emergence of untreatable ceftriaxone-resistant NG strains in the future, given that this drug is the most effective antimicrobial available in treating NG infection. This high prevalence of NG treated with ceftriaxone with an MIC of 0.125 mg/L or higher in China exceeds that in other countries.26–28 This confirms the necessity of increasing the currently recommended dose of 250-mg ceftriaxone for treating NG infection in China, at least in those 3 cities. Another problem in China is that a fair number of STD doctors deem that the ceftriaxone made in China may be less effective than that made in the United States, so they use 2 g or a higher dose of the ceftriaxone made in China to treat NG infection to ensure its efficacy. In the future, it is necessary to do clinical trials to compare the ceftriaxone made in China with that made in the United States in efficacy in treating NG infection.

In fact, cefixime has never been recommended as first-line treatment for NG infection in China. The mean prevalence of NG susceptible to cefixime in the 3 cities was only 74.6%, notably lower than a 95% efficacy threshold; according to the guidelines established by the STD CDC of China in 2013, this drug should remain excluded from first-line treatment for NG infection in China.6 Coincidently, cefixime is not a first-line drug either for treating NG infection in Japan, the United States, and European countries.2–5

Among all isolates gathered in the 3 cities, those from Guangzhou had the highest prevalence of TRNG, NG treated with ceftriaxone with an MIC of 0.125 mg/L or higher, and NG nonsusceptible to cefixime. This indicated that NG isolates from Guangzhou were more prone to develop resistance to ceftriaxone, cefixime, and tetracycline than those from Nanjing and Tianjin. This was probably related to some features of Guangzhou: it is near the city of Dongguan (also in Guangdong Province), which is commonly known as a sexual hub with rampant commercial sexual services, and its economy is booming, which benefits from Hong Kong's economic prosperity. Currently, Chinese epidemiologists are seriously concerned that gonorrhea might become untreatable under some circumstances, especially in some areas in China. To contain the development of ceftriaxone resistance by NG isolates, the National Center for STD Control, China CDC, and pharmaceutical companies need to make efforts in the following 3 aspects. First, pay more attention to pharyngeal NG infection in men who have sex with men and the female population, given that NG isolates obtained from these populations have been demonstrated to be prone to develop ceftriaxone resistance.17 Second, in light of ceftriaxone given alone in China but in combination with azithromycin in other countries in the world for treating NG infection, it is warranted to conduct clinical trials to compare the 2 different prescribing practices with respect to efficacy. Third, it is urgent to develop NG vaccine and antimicrobials, which is expected to play a significant role in militating against this infection.

To investigate the genetic relatedness of nonsusceptible NG isolates, in this study, we performed MLVA on all of the 244 isolates by following the protocol of Kushnir et al15,16 instead of that by Heymans et al17–20 because the former costs less than the latter, and a cost-effective molecular genotyping method is certainly favorable in developing countries like China. However, the former does require more hands-on time and was not a DNA sequencing method. In addition, one of the most important disadvantages was subjective interpretation of results. A total of 110 MLVA types were identified among the 244 NG isolates. Multiple-locus variable number tandem repeat analysis type 25 was the most prevalent and present in 7.14% (5/70) of isolates from Guangzhou, 8.74% (9/103) of isolates from Nanjing, and 16.90% (12/71) of isolates from Tianjin, which suggested that this specific MLVA type had a high transmission level.

We initially hypothesized that patients infected with NG isolates with similar genotypes were likely to be epidemiologically linked. To test this hypothesis, a minimum spanning tree was constructed according to MLVA profiles. Clustering yielded 5 clusters of 13 to 85 members, including 241 of all 244 isolates (Fig. 1). Cluster III, in which 50.6% of isolates were from Nanjing, was associated with PPNG, implicating that NG isolates from Nanjing might be prone to evolve into PPNG. Consistently, it has been reported that Nanjing had higher PPNG prevalence than other cities.29,30 In this study, isolates in cluster II, all recovered from males, were associated with non-PPNG, NG with decreased ceftriaxone susceptibility, and antibiotic use. This implies that NG-infected males who used antibiotics are at a high risk for infection with NG with decreased ceftriaxone susceptibility, so they should be warned of this risk. On the other side, the 5 clusters did not significantly differ in the prevalence of NG nonsusceptible to cefixime, which can be explained by the fact that this drug has not been used as first-line treatment for NG infection in China.

The present study has some limitations: (1) all tested NG isolates were recovered from STD clinic attendees; such a population very likely had higher prevalence of antimicrobial resistance than the general population, and they could not represent the 3 cities well; (2) this study had different numbers of isolates collected from the 3 cities, which could reduce the statistical power; (3) the breakpoints we used in this study were different for ceftriaxone and cefixime, which may lead to difficulty in comparing the susceptibility of ceftriaxone and cefixime with other studies; and (4) it was difficult to compare the results of this study with that of other studies for the subjective judgment of the method used in this study.

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CONCLUSIONS

Successful antibiotic treatment is essential for controlling NG infection. Hence, many initiatives have been directed at promoting and sustaining national and international NG antimicrobial susceptibility surveillance as a key measure to promptly inform the public and update guidelines for treatment of this infection. Our results confirm that at present, penicillin, tetracycline, ciprofloxacin, and cefixime are not suitable for treating NG infection in China, but ceftriaxone and spectinomycin remain effective in this respect. In addition, the MLVA scheme used in this study was successful in typing NG isolates, resulting in 5 clusters, and some clusters were associated with PPNG and NG with decreased ceftriaxone susceptibility.

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