A total of 132 blood samples were collected from female patients from the Yongkang region with primary syphilis in The First People's Hospital of Yongkang (Zhejiang, China) between May 2016 and June 2017. Patients with HIV or T. pallidum history were excluded from the present study. The protocols were approved by the Human Subjects Division of The First People's Hospital of Yongkang. All patients were required to provide written informed consent. T. pallidum strains were isolated from patient blood samples using sequencing-based typing or enhanced Centers for Disease Control and Prevention typing as described previously (17). DNA samples were tested for T. pallidum using quantitative polymerase chain reaction (PCR) targeting the polA gene (18).

T. pallidum DNA was isolated as previously described (19). Briefly, DNA was extracted from 200 µl of whole blood and DNAzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to extract DNA from specimens, according to the manufacturer's protocols. Strain typing was classified based on the analysis of three DNA target regions: (1) Restriction fragment length polymorphism analysis of sequence differences in the tpr gene; (2) the number of 60 bp repeats in the arp gene; (3) sequence analysis of a short region of the tp0548 gene (20).

The PCR assay for T. pallidum analyzed the gene target polA as described previously (21). The primers used were as follows: F: 5′-CGTGTGGTATCAACTATGG-3′, R: 5′-TCAACCGTGTACTCAGTGC-3′. All PCR products were analyzed using an ABI9700 GeneAmp PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was performed in a 25 µl reaction volume with 0.1 µg DNA, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.5 µM primers and 0.5 µl Taq polymerase (Takara Bio, Inc., Otsu, Japan). PCR conditions were as follows: 96°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 56°C for 56 sec and 72°C for 60 sec, followed by 72°C for 600 sec. PCR products were analyzed on a 1.5% agarose gel. Then, PCR products were visualized by staining with ethidium bromide and comparing with the molecular size markers of a 100- or 2,000-bp ladder (New England BioLabs, Inc., Ipswich, MA, USA).

The polA gene was amplified to detect resistance to azithromycin (22). Briefly, PCR amplification of the 23S rRNA gene of T. pallidum was treated by restriction enzyme digestion (MboII) as described previously (23,24). PCR were performed in a 50 µl reaction volume with 0.2 µg rRNA, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.5 µM primers (5′-GTGCCAGCMGCCGCGG-3′) and 1.0 µl Taq polymerase. PCR conditions were as follows: 95°C for 5 min, followed by 35 cycles at 94°C for 30 sec, 54°C for 57 sec and 72°C for 60 sec, followed by 72°C for 600 sec. The azithromycin resistance genotypes were analyzed by DNA sequencing of the PCR products after purification with a QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA, USA). T. pallidum DNA was also evaluated using restriction enzyme digestion for A2058G and A2059G mutations (MboII and BsaI, respectively; New England BioLabs, Inc.) (25). The DNA sequences were obtained using the DNA analyzer 3730×l (Applied Biosystems; Thermo Fisher Scientific, Inc.) and analyzed using DNASTAR^® software version 3.0 (DNASTAR Inc., Madison, WI, USA).

All antibiotic disks (azithromycin) were purchased from Abtek Biologicals Ltd. (Liverpool, UK). An antibiogram was performed on Mueller-Hinton agar for 12 h at 37°C to determine the antimicrobial agents resistance profiles to azithromycin (10 µg) or PBS (10 µg). Antimicrobial susceptibility was performed according to the European Committee on Antimicrobial Susceptibility guidelines (26).

Data are expressed as mean ± standard deviation of triplicate experiments. All data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Differences among groups were analyzed by one-way analysis of variance with Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

DNA samples from 132 genital ulcer specimens were identified as T. pallidum positive, determined by PCR targeting the polA gene (Fig. 1A). Three gene types were observed among the 132 T. pallidum positive specimens (Fig. 1B). The restriction digestion assay indicated that 78 samples were tpr gene type, 24 were arp gene type and 30 were tp0548 gene type. These results suggest that tpr is the most common gene type of T. pallidum.

The antibiotic resistance of T. pallidum to azithromycin was analyzed in this study. As indicated in Fig. 2A, the restriction fragment length polymorphism analysis of the PCR amplicons from the representative samples (A2058G: 14a/f, 14e/f and 12e/f; A2059G: 8d/f, 12d/f, 6d/f, 11d/f, 14j/f.) revealed that 94 of the T. pallidum specimens were resistant to azithromycin. Gene type analysis indicated that the tpr gene type presented significantly higher drug resistance compared with the arp and tp0548 gene types (Fig. 2B). These results suggest that tpr gene type may be associated with drug resistance of T. pallidum to azithromycin.

The association between gene subtype tpr of T. pallidum and drug resistance was evaluated further. Eight subtypes of tpr (14a/f, 14e/f, 12e/f, 12d/f, 6d/f, 11d/f, 14j/f, 8d/f) were identified among the 132 cases (Table I). It was identified that 23S rRNA A2058G mutation was observed in gene subtypes 14a/f, 14e/f and 12e/f. A2059G mutation occurred in gene subtypes 8d/f, 12d/f, 6d/f, 11d/f and 14j/f. The proportion of azithromycin-resistant genotypes harboring either the A2058G or the A2059G mutation among T. pallidum was 65.8 and 34.2%, respectively (Fig. 3). These results indicate that tpr gene subtypes of T. pallidum may be associated with drug resistance to azithromycin.

The drug resistance of A2058G and A2059G mutations in gene subtype tpr of T. pallidum was investigated further. As indicated in Fig. 4A, A2059G mutation demonstrated a higher drug resistance for azithromycin compared with A2058G mutation, as determined by antimicrobial susceptibility test. The distribution of gene subtypes of T. pallidum with A2059G and A2058G mutation is presented in Fig. 4B and C. These results demonstrated that 14a/f of A2058G mutation and 12d/f of A2059G mutation frequently occurred in tpr of T. pallidum. These results indicate that A2058G and A2059G mutations in gene subtype tpr of T. pallidum are associated with drug resistance to azithromycin.