Gonorrhea, caused by Neisseria gonorrhoeae, has become one of most prevalent sexually transmitted infections, with a global estimated incidence of 106.1 million infected people annually in 2008.1 Control of gonorrhea relies on comprehensive strategies including appropriate treatment with antimicrobials. However, N. gonorrhoeae has shown an extraordinary capacity to develop resistance to essentially all antimicrobials introduced as first-line treatment.2–4 In regard to penicillin, the first plasmid-mediated high-level penicillin-resistant strains, that is, the penicillinase-producing N. gonorrhoeae (PPNG) isolates, were identified in 1976 and have been subsequently spreading worldwide.5,6 Recently, the emerging threat of untreatable gonococcal infection caused by chromosomally mediated high-level resistance to ceftriaxone has been warned in some developed countries.4,7–9 However, such threat may be even bigger in developing countries where the surveillance program has not been well established, but antibiotics are widely used (or misused sometimes) in medical practice.10,11
In Enterobacteriaceae, plasmid-carried blaTEM gene variants that encode extended-spectrum β-lactamases (ESBLs) have been described, and it is reported that these ESBLs, which might degrade extended-spectrum cephalosporins including ceftriaxone, usually result from acquisition of relatively few specific mutations in the blaTEM gene.12 Compared with blaTEM-1, blaTEM-135 has a single amino acid substitution at position 182, where methionine has been substituted by threonine (M182T).13 This amino acid position is not included in any active site motif of the β-lactamase, but it is located in the hinge region between 2 important domains. The role of this M182T amino acid substitution has been proposed to include a stabilization of the active site topology reorganized by other mutations, which collaboratively results in the emergence of a stable ESBL.14 Although ESBL has not been reported in N. gonorrhoeae, PPNG isolates possessing a blaTEM-135 gene have been found in some Asian countries such as Japan and Thailand.15,16 However, epidemiological data of PPNG strains possessing blaTEM-135 gene are not available in China. In the present study, we preliminarily investigate the prevalence of PPNG and blaTEM-135 and molecular analyses among the clinical gonococcal isolates collected in 2007 and 2012 in Nanjing, China.
MATERIALS AND METHODS
N. gonorrhoeae Isolates and PPNG Testing
A total of 199 and 77 N. gonorrhoeae isolates were consecutively collected from patients with urogenital gonorrhea attending clinic at the National Center for STD Control, Nanjing, China, in 2007 and 2012, respectively. This clinic is one of the sentinel clinics participating in the National Gonococcal Resistance Program in China and captures most of sexually transmitted disease (STD) cases in the study area. Female endocervical swabs and male urethral swabs were used for sample collection. After isolation using selective culture and subsequent N. gonorrhoeae species confirmation using oxidase reaction and carbohydrate use tests, nitrocefin tests were performed to identify PPNG.17 All isolates were stored in skim milk at −80°C.
DNA was extracted from bacterial suspensions using QIAxtractor DX Kits (Qiagen, Hilden, Germany) on a QIAxtractor automated genomic DNA extraction instrument, according to the manufacturer’s instructions (Qiagen).
Identification of blaTEM-135 Using Mismatch Amplification Mutation Assay Polymerase Chain Reaction
Mismatch amplification mutation assay (MAMA) polymerase chain reaction (PCR) was performed, as previously described,16 to identify blaTEM-135 among all PPNG isolates. Briefly, the primers TEM-F (5′-GTCGCCCTTATTCCCTTTTTTG-3′) and TEM-R (5′-TAGTGTATGCGGCGACCGAG-3′) amplified a segment of both the blaTEM-1 and blaTEM-135 genes, and the primers MAMA-F (5′-GCATCTTACGGATGGCATGAC-3′) and MAMA-R (5′-TGTTGCCATTGCTGCAGGGG-3′) amplified a fragment of the blaTEM-135 gene only. All primers were synthesized by Invitrogen, Beijing, China. All PCRs were performed by using an GeneAmp PCR 9700 Thermocycler (Applied Biosystems), with incubation at 96°C for 2 minutes, followed by 25 cycles of denaturation at 96°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 30 seconds. Final extension at 72°C for 2 minutes was performed. NGON 00-002 or NGON 00-027 (containing blaTEM-1), and NGON04-025 or NGON08-003 (containing blaTEM-135) were used as controls in all PCRs. Polymerase chain reaction products were identified in a 1.5% agarose (Oxoid, Hampshire, England, UK) gel electrophoresis. The whole blaTEM gene was amplified and sequenced at BGI (Shenzhen, China), as previously described,15 in random isolates (n = 8), to confirm the blaTEM-135 and that no additional mutations existed in the blaTEM-135 gene.
Molecular Epidemiological Examinations
N. gonorrhoeae multiantigen sequence typing analysis was performed as described previously.18 To investigate the phylogenetic relationship of the PPNG isolates possessing blaTEM-135, a phylogenetic tree was constructed based on partial porB gene sequences (490 base pairs) in the isolates possessing blaTEM-135 from the current study and compared them with those from the previous reports in Japan and Thailand. PorB sequences of Thailand and Japan isolates were downloaded from http://www.ng-mast.net Web site, according to NG-MAST STs in the references.15,16 The software BioEdit version 7.1.9 and MEGA5 (neighbor-joining algorithm) were used for the phylogenetic analysis as earlier depicted.19
The prevalence with 95% confidence intervals (CIs) was measured. Associations between categorical variables were determined by the χ 2 test.
Prevalence of PPNG and blaTEM-135
A total of 276 patients attending the sexually transmitted disease clinic were included in the analyses in 2007 and 2012. Of the 276 patients ranging in age from 15 to 85 years (mean ± SD, 36.4 ± 11.0 years), 230 (83.3%) were male. In 2007, 90 isolates (45.2%; 95% CI, 38.5–52.2%) produced β-lactamase (PPNG isolates), among which 52 (57.8%; 95% CI, 47.5–67.5%) possessed blaTEM-135 (blaTEM-135–possessing isolates). The prevalence of PPNG isolates decreased to 31.2% (95% CI, 21.9–42.2; P = 0.05) in 2012, but the proportion of blaTEM-135–possessing isolates remained at a high level of 58.3% (95% CI, 24.5%–61.2%). The prevalence of PPNG was significantly higher among older patients (age > 36 years) than among younger patients (χ 2 = 4.8, P = 0.03). No significant differences of blaTEM-135–possessing proportion were observed between different age groups or different sexes.
NG-MAST Analysis of N. gonorrhoeae Isolates
In total, 162 NG-MAST STs (122 STs in 2007 and 48 STs in 2012) were identified among the 276 isolates, of which 73 STs (54 STs in 2007 and 27 STs in 2012) were those identified in previous studies and 89 STs (68 STs in 2007 and 21 STs in 2012) were first identified from the current study. ST568 (n = 8) was the most common ST, followed by ST270 (n = 5), ST2288 (n = 4), ST421 (n = 4), ST8770 (n = 4), and ST8726 (n = 4). Eight STs (ST270, ST436, ST568, ST1742, ST1766, ST1866, ST3284, and ST3356) were simultaneously identified in isolates from 2007 and 2012. The PPNG isolates (n = 114) and blaTEM-135–possessing isolates (n = 66) were divided into 74 and 38 STs, respectively (Table 1). Among the PPNG isolates, a potential association between specific STs and presence of blaTEM-135 was found. For instance, all isolates with ST270, ST1053, ST1866, ST1868, ST2288, ST2318, ST8725, ST8726, and ST8770 possessed blaTEM-135. Notably, some STs such as ST421, ST568, ST1742, and ST1766 could possess blaTEM-135 or non–blaTEM-135 variant (presumably blaTEM-1).
Phylogenetic Analysis of blaTEM-135 Variants
The results from phylogenetic analysis of current PPNG isolates possessing blaTEM-135 and its comparison with those from Japan and Thailand are shown in Figure 1. The isolates were divided into 2 main clades (A and B), which represented the 2 different serogroups PorB1b (WII/III) and PorB1a (WI), respectively, of N. gonorrhoeae. All isolates from the current study (n = 18) in the smaller clade B (PorB1a isolates) were from 2007, whereas clade A (PorB1b isolates) were from 2012 (n = 14) and a large proportion (n = 33) of the Chinese isolates were from 2007. The previously identified blaTEM-135–possessing isolates from Japan and Thailand were spread in both clades, and some of those, that is, ST4013-Japan and ST211-Thailand, contained an identical porB gene sequence as some of the Chinese blaTEM-135-possessing isolates (Fig. 1).
Gonorrhea and its control are a major public health issue in the study area. This study was conducted in a city along the coastal areas where sexually transmitted infections are mostly prevalent in China.20 According to the data from the national surveillance system, 2129 and 810 cases with gonorrhea were reported from Nanjing in 2007 and 2012, respectively. Although a decreasing trend was observed over the period, it is generally believed that underreporting caused by patients’ self-treatment with antibiotics is a big concern that not only compromises the surveillance program but also increases selective pressure to facilitate the development of drug resistance.
The prevalence of PPNG was less than 10% in the 1990s, but then increased dramatically in recent 2 decades. The overall prevalence in China reached a peak of 45.2% in 2007.21–23 In addition to high prevalence of PPNG among clinical isolates, high proportions of blaTEM-135 genes in the PPNG isolates over the years (2007 and 2012) were found in the current study. Although the prevalence of PPNG (43.1%) is lower than that observed in some Asian countries, the proportion of blaTEM-135 (57.9%) in the current study is substantially higher than that in Japan or Thailand. Because blaTEM-135 only requires one additional specific mutation to evolve into an ESBL, such as blaTEM-20, that could degrade also ceftriaxone, the current findings may potentially imply an emerging threat of untreatable gonococcal infection in China. In consideration of this potential threat, it may be necessary to include periodic surveys of blaTEM-135 and other blaTEM variants, as an early warning indicator, in the current gonococcal resistance surveillance program of gonococcal resistance.
A total of 162 NG-MAST STs, of which 89 STs were novel, were found in the present study, suggesting that the diversity of N. gonorrhoeae strains transmitted in Nanjing is high. The high number of new STs probably represents that very few N. gonorrhoeae isolates from China have been previously investigated with NG-MAST and many locally emerged STs are circulating in this region. In previous studies of blaTEM-135–possessing isolates in other Asian countries, ST1549 and ST4013 were found in Japan, whereas ST5134, ST5138, ST5132, ST1292, ST147, ST5135, and ST211 were found in Thailand.15,16 None of those STs were found in Nanjing, which suggests that many different N. gonorrhoeae blaTEM-135 strains have evolved separately in these countries. Nevertheless, a phylogenetic tree of partial porB gene sequences (490 base pairs) showed that some of the previously identified blaTEM-135–possessing isolates from Japan15 and Thailand,16 that is, ST4013-Japan and ST211-Thailand, contained an identical porB gene sequence as some of the Chinese blaTEM-135–possessing isolates, indicating a common evolutionary origin and possibly some overlap of the sexual networks in those countries.
Penicillinase-producing N. gonorrhoeae have been associated with specific auxotype and serovar classes (A/S) in previous study.24 In our present study, a strong association between specific NG-MAST STs and the presence of blaTEM-135 was also found. In many studies worldwide, associations between specific NG-MAST STs and antimicrobial resistance phenotypes and genotypes have been described. For example, the ST1407, as well as its evolved genetic subtypes, has been identified as a mainly globally spread ST that has accounted for a high proportion of the decreased susceptibility or resistance to extended-spectrum cephalosporins in many countries.4,8
The current study has some limitations to be addressed. First, although the isolates used for the current study can be the representative clinic-based samples from the study area because they were collected during the whole year in the clinic, which captured most patients seeking for services, any generalization of the results from this study to other areas in China or to those patients who seek self-treatment should be made with caution. Second, the current study was conducted in 2 years; it may be hard to really describe the trend of blaTEM-135 containing strains prevalence over time. Third, more clinical data including the previous use of antibiotics and the treatment of the gonococcal infection were not systematically collected, resulting in a poor analysis of their associations with the occurrence of blaTEM-135 containing strains. Despite these limitations, the current study may be helpful for highlighting the issue of blaTEM-135 containing strains and planning for surveillance program and relevant studies in future in China.
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