Markedly Increasing Antibiotic Resistance and Dual Treatment of Neisseria gonorrhoeae Isolates in Guangdong, China, from 2013 to 2020

ABSTRACT

The emergence of multidrug resistance in Neisseria gonorrhoeae is concerning, especially the cooccurrence of azithromycin resistance and decreased susceptibility to extended-spectrum cephalosporin. This study aimed to confirm the antibiotic resistance trends and provide a solution for N. gonorrhoeae treatment in Guangdong, China. A total of 5,808 strains were collected for assessment of antibiotic MICs. High resistance to penicillin (53.80 to 82%), tetracycline (88.30 to 100%), ciprofloxacin (96 to 99.8%), cefixime (6.81 to 46%), and azithromycin (8.60 to 20.03%) was observed. Remarkably, spectinomycin and ceftriaxone seemed to be the effective choices, with resistance rates of 0 to 7.63% and 2.00 to 16.18%, respectively. Moreover, the rates of azithromycin resistance combined with decreased susceptibility to ceftriaxone and cefixime reached 9.28% and 8.64%, respectively. Furthermore, genotyping identified NG-STAR-ST501, NG-MAST-ST2268, and MLST-ST7363 as the sequence types among representative multidrug-resistant isolates. Evolutionary analysis showed that FC428-related clones have spread to Guangdong, China, which might be a cause of the rapid increase in extended-spectrum cephalosporin resistance currently. Among these strains, the prevalence of N. gonorrhoeae was extremely high, and single-dose ceftriaxone treatment might be a challenge in the future. To partially relieve the treatment pressure, a susceptibility test for susceptibility to azithromycin plus extended-spectrum cephalosporin dual therapy was performed. The results showed that all the representative isolates could be effectively killed with the coadministration of less than 1 mg/liter azithromycin and 0.125 mg/liter extended-spectrum cephalosporin, with a synergistic effect according to a fractional inhibitory concentration (FIC) of <0.5. In conclusion, dual therapy might be a powerful measure to treat refractory N. gonorrhoeae in the context of increasing antibiotic resistance in Guangdong, China.

INTRODUCTION

Gonorrhea, caused by Neisseria gonorrhoeae, remains one of the most common sexually transmitted diseases worldwide. Bacteria infecting the genitourinary system, rectum, and oropharyngeal mucosa can cause urethritis and cervicitis and increase susceptibility to and transmission of HIV (1). The World Health Organization (WHO) estimated a prevalence of approximately 82.4 million cases annually, a 54.4% increase since 2014 (2). Among these cases, patients in China accounted for a large proportion, with approximately 115,000 new cases per year (3). N. gonorrhoeae transmission may be associated with the continuous increase in antimicrobial resistance (4); thus, strengthening drug resistance surveillance is essential for curbing the epidemic and adjusting the clinical guidelines for N. gonorrhoeae.

Antibiotics, such as penicillin (PEN), ciprofloxacin (CIP), tetracycline (TET), azithromycin (AZM), cefixime (CFM), and ceftriaxone (CRO), had been effective for N. gonorrhoeae treatment until the emergence of multidrug-resistant (MDR) strains. Currently, only CRO is recommended as a first-line monotherapy in China. As reported, >1-g CRO therapy is effective in uncomplicated gonorrhea (5), but a high dose also causes severe damage to the liver (6) and is a threat to health, the environment, and antimicrobial resistance (7). Currently, the increase in strains with decreased susceptibility to CRO (CRO^d) (8), especially the internationally distributed high-level CRO-resistant FC428 clone (penA-60.001) (9), has reduced the clinical effectiveness of CRO. To curb the spread of CRO resistance and relieve treatment pressure before the application of new antibiotics, dual-therapy AZM plus extended-spectrum cephalosporin (ESC) has been proposed (10). However, with the increase in AZM-resistant (AZM^r) strains, especially clones with high-level resistance (MIC ≥ 256 mg/liter) isolated in China (11), Australia (12), England (13), Japan (14), Canada (15), and Denmark (16), and the first reported combination therapy clinical failure in the United Kingdom (17), the dual-therapy recommendations are controversial. Currently, whether this approach is effective in the context of cooccurrence of decreased cephalosporin susceptibility (ESC^d) and AZM^r remains unclear.

Guangdong Province, with a population of more than 100 million, has been the epicenter of gonococcal infection in China. In this study, we clarified the severity of drug resistance in this province by determining the antimicrobial susceptibility of 5,808 isolates and then genotyped NG-STAR-ST501, NG-MAST-ST2268, and MLST-ST7363 as the main sequence types (STs). Notably, some strains homologous to the highly CRO-resistant clone FC428 have spread to Guangdong, China, which might further increase the burden on N. gonorrhoeae treatment. Here, we assessed the feasibility of dual therapy against MDR N. gonorrhoeae, and the results showed that the drug combination had a strong synergistic action with a fractional inhibitory concentration (FIC) of <0.5 in vitro. Thus, in the context of increasing antibiotic resistance, dual therapy might be an effective alternative before the application of new drugs.

RESULTS

Antimicrobial resistance trends of N. gonorrhoeae in Guangdong, China (2013 to 2020).

To clarify the antibiotic resistance trends among N. gonorrhoeae strains and provide a basis for clinical treatment guidelines, 5,808 strains were collected for MIC testing. As shown in Fig. 1A, the PEN resistance rate fluctuated from 53.80% to 82.00%, and almost all strains were resistant to TET (88.30 to 100%) and CIP (96.00 to 99.80%). In addition, the MIC distribution ratios of the above-described antibiotics by year showed small differences (Fig. 1B to D). For SPT, the resistance rate reached 7.63% in 2019 but dropped to 0.25% the following year (Fig. 1A). Furthermore, the distribution ratios of SPT resistance showed that drug-resistant strains with a MIC of ≥128 mg/liter began to appear in 2019 (Fig. 1E). Remarkably, the decreased rate of susceptibility to CRO has been higher than 5% in recent years (Fig. 1A), reaching 16.18% (18), and the first high-level CRO-resistant isolate (MIC = 0.5 mg/liter) spread here in 2016 (Fig. 1F). For CFM, we noted clones with decreased susceptibility in all years, accompanied by a rate of decreased susceptibility of 6.00 to 23.16% (Fig. 1A). In particular, we generally observed the highest proportion (MIC of ≥1 mg/liter) of ESC-resistant isolates in 2019 (Fig. 1G). Finally, resistance surveillance data showed an increase (from 8.60% to 20.03%) in the proportion of AZM^r N. gonorrhoeae strains between 2013 and 2018, followed by a decrease from 2019 (Fig. 1A). As shown, the distribution revealed that the proportion of isolates with a MIC of  ≥2 mg/liter had increased, and many highly resistant strains with a MIC of  ≥8 mg/liter were identified (Fig. 1H). In conclusion, antibiotic resistance in Guangdong, China, is extremely high.

FIG 1.

Antimicrobial resistance trends of N. gonorrhoeae isolates from Guangdong, China. (A) Percentage of gonococcal isolates resistant to PEN, TET, CIP, SPT, and AZM and with decreased susceptibility to CRO and CFM. (B to H) Proportion of N. gonorrhoeae isolates with different MICs (mg/liter) for PEN (B), TET (C), CIP (D), SPT (E), CRO (F), CFM (G), and AZM (H) (n = 5,808 isolates). (I) Percentage of gonococcal isolates with both AZM^r and CRO^d (solid circle) or CFM^d (blank circle). PEN, penicillin; TET, tetracycline; CIP, ciprofloxacin; SPT, spectinomycin; AZM, azithromycin; CFM, cefixime; CRO, ceftriaxone; AZM^r, azithromycin resistance; CRO^d, decreased susceptibility to ceftriaxone; CFM^d, decreased susceptibility to cefixime.

Since CRO, CFM, and AZM were candidates for dual therapy, we further analyzed the frequency of both AZM^r and ESC^d. As shown in Fig. 1I, the proportion of AZM^r and CRO^d isolates reached 9.28% in 2019, and the proportion of AZM^r and CFM^d dual-resistant strains was 8.64%. Although the rates decreased in 2020, the severity of antibiotic resistance in Guangdong, China, remains evident.

Genotyping and evolutionary analysis of MDR N. gonorrhoeae.

To analyze the molecular epidemiology of MDR isolates, 79 strains were selected and genotyped by NG-STAR, NG-MAST, and MLST. According to NG-STAR (Fig. 2A and Table S1A in the supplemental material), 20 new STs were defined for the first time, and a total of 27.5% of known STs were shared by ≥2 isolates, including ST501 (n = 6), ST345/ST348/ST428/ST1656 (n = 3), and ST495/ST497/ST929/ST1463/ST1687/ST2473 (n = 2). Moreover, most were related to AZM^r and ESC^d (Table S2). Unexpectedly, a high-level CRO- and AZM-resistant isolate named 1669, possessing the penA-60.001 allele, was identified in Guangdong, China (Tables S1A and S2). However, the penA-18.001 nonmosaic and penA-10.001 mosaic alleles were more widely distributed in this area (Fig. 2B). According to NG-MAST (Fig. 2C and Table S1B), the strains could be grouped into 35 known STs, covering 44.3% of the 79 strains, and 34 new genotypes were identified. Among them, the ST2268 (n = 4), ST10209 (n = 3), and ST304/ST1766/ST1866/ST5062 (n = 2) alleles were identified with a prevalence of >4.4% (n ≥ 2). Finally, each isolate could be assigned to a specific ST by MLST, except for the strain named SG19 (Table S1C). For MLST, we identified 35 STs, with ST7363 (n = 9) being the most prevalent, followed by ST1901 and ST1928 (n = 7) (Fig. 2D).

FIG 2.

Genotyping and evolutionary analysis of MDR N. gonorrhoeae. (A) The number of each sequence type was counted according to NG-STAR. (B) The number of each penA allele carrying a nonmosaic or mosaic gene. Amino acid alterations within the mosaic penA allele related to decreased susceptibility to ESC are cited in red. (C and D) The number of each sequence type was counted according to NG-MAST and MLST analyses. The colored histogram indicates ≥2 ST. NG-STAR, N. gonorrhoeae sequence typing for antimicrobial resistance; NG-MAST, N. gonorrhoeae multiantigen sequence typing; MLST, multilocus sequence typing; NA, new allele. (E) Multilocus phylogeny of 79 MDR isolates and two identified FC428 clones (FC428 and GC249). The phylogenetic tree was constructed based on MLST alleles. Bootstrap values higher than 85% are indicated. Red font indicates the original FC428 strain found in Japan, and purple font indicates the FC428-related clone GC249 identified in China. MLST, multilocus sequence typing; MDR, multidrug resistant.

Furthermore, to confirm the origin and transmission, the sequences in the subclones identified by MLST genotyping were subjected to evolutionary analysis. According to Fig. 2E, strains 1669 and ST31 (blue) were clustered with the globally disseminated CRO^r FC428 origin clone (red) (19). In addition, the local strains SL3 and SS34 (green) and FC428-related clone GC249 (purple) (20), found in Changsha, China, had a relatively high degree of homology. The other subclones were classified into different clusters due to genetic diversity. Overall, FC428-related clones have spread across Guangdong, China; thus, it is imperative to strengthen molecular monitoring.

Evaluation of the efficacy of dual therapy against N. gonorrhoeae.

To evaluate the effectiveness of dual therapy and offer a solution to the threat of antibiotic resistance in Guangdong, China, representative MDR strains were used for assessment. As shown in Table 1, all strains showed susceptibility to less than 1 mg/liter AZM combined with 0.125 mg/liter CFM, with the highest MIC reductions of 4- to 167-fold and 3- to 250-fold being observed for AZM and CFM in combination therapy. The median MIC of AZM decreased from 3.2 ± 3.098 mg/liter to 0.221 ± 0.236 mg/liter, and that of CFM decreased from 0.256 ± 0.217 mg/liter to 0.03 ± 0.03 mg/liter (see Table 3). Under dual therapy with AZM and CRO, the effectiveness of the medicines increased 4- to 267-fold for AZM and 4- to 62-fold for CRO (Table 2). The mean MIC of AZM decreased from 2.77 ± 3.02 mg/liter when used alone to 0.20 ± 0.23 mg/liter in the combination treatment, and the corresponding decrease for CRO was from 0.16 ± 0.09 mg/liter to 0.026 ± 0.027 mg/liter (Table 3). Importantly, ESC^d strains with cooccurring high-level AZM^r (MIC of ≥8 mg/liter) could be killed effectively with lower antibiotic concentrations within the susceptibility breakpoint. Finally, an FIC of <0.5 confirmed that dual therapy resulted in a synergistic effect for the inhibition of bacterial growth inhibition in vitro, and lower concentrations could be used in the combination regimen to effectively treat N. gonorrhoeae infections (Fig. 3). Overall, dual therapy might still be an effective solution for resolving N. gonorrhoeae infections and addressing the prevalence of N. gonorrhoeae in the context of high antibiotic resistance.

TABLE 1.

Determination of MIC (mg/liter) of AZM and CFM alone or in combination against MDR gonorrhoeae isolates^a

Strain	MICalone		MICCombination		FCalone/comb
	AZM	CFM	AZM	CFM	AZM	CFM
1516	≥8	0.125	0.25	0.015	≥29	8
1525	4	0.125	0.25	0.004	16	31
1526	1	0.25	0.03	0.03	33	8
1531	1	0.25	0.03	0.008	33	31
1538	≥8	0.25	0.25	0.015	≥32	17
1690	≥8	≥1	1	0.06	≥8	≥17
1668	2	0.125	0.06	0.004	33	31
1547	≥8	0.125	0.25	0.015	≥32	8
1618	1	0.25	0.03	0.06	33	4
1692	1	0.125	0.03	0.015	33	8
1738	4	0.25	0.03	0.06	133	4
1669	1	≥1	0.125	0.004	8	≥250
1678	1	0.125	0.03	0.02	33	6
1647	1	0.25	0.03	0.03	33	8
1540	1	0.125	0.06	0.004	17	31
1549	≥8	0.5	0.25	0.002	≥32	250
1583	1	0.125	0.03	0.002	33	62
1569	≥8	0.125	0.25	0.015	≥32	8
1565	1	0.125	0.03	0.002	33	63
1338	1	0.25	0.25	0.06	4	4
1362	≥8	0.5	0.5	0.125	≥16	4
1353	1	0.125	0.03	0.002	33	63
1355	1	0.5	0.03	0.002	33	250
SS6	1	0.125	0.125	0.008	8	16
SS11	1	0.125	0.125	0.008	8	16
SS12	1	0.25	0.25	0.03	4	8
SS30	2	0.125	0.5	0.03	4	4
SS34	1	0.125	0.125	0.015	8	8
SS36	1	0.25	0.006	0.03	167	8
SS37	≥8	0.125	0.25	0.04	≥32	3
SS38	1	0.125	0.125	0.015	8	8
SS39	1	0.5	0.125	0.03	8	17
SS42	4	0.125	0.5	0.03	8	4
SS43	>8	0.5	1	0.125	>8	4
SS53	1	0.25	0.25	0.004	4	63
SS55	1	0.125	0.25	0.004	4	31
SS72	1	0.125	0.125	0.015	8	8
SS73	1	0.5	0.25	0.06	4	8
SS76	≥8	0.125	0.5	0.015	16	8
SS78	>8	0.125	0.5	0.03	>16	4

^aFC, fold change; Comb, antibiotics combination.

TABLE 3.

MIC of each antimicrobial (alone and in combination) against MDR gonorrhoeae isolates

No. of isolates	MIC (median ± SD, mg/liter)
	AZM alone	AZM in combination with ESC	ESC alone	ESC in combination with AZM
40 (CFM&AZM)	3.2 ± 3.098	0.221 ± 0.236	0.256 ± 0.217	0.03 ± 0.03
21 (CRO&AZM)	2.77 ± 3.02	0.20 ± 0.23	0.16 ± 0.09	0.026 ± 0.027

TABLE 2.

Determination of MIC (mg/liter) of AZM and CRO alone or in combination against MDR gonorrhoeae isolates^a

Strain	MICalone		MICCombination		FCalone/comb
	AZM	CRO	AZM	CRO	AZM	CRO
1690	≥8	0.125	0.03	0.015	≥267	8
1729	1	0.125	0.03	0.004	33	31
1669	1	0.25	0.125	0.004	8	62
1647	1	0.125	0.03	0.03	33	4
1531	1	0.125	0.03	0.004	33	31
1538	≥8	0.125	0.03	0.015	≥267	8
SS2	2	0.125	0.25	0.008	8	16
SS12	1	0.125	0.25	0.015	4	8
SS13	1	0.125	0.25	0.015	4	8
SS14	1	0.125	0.25	0.015	4	8
SS15	1	0.125	0.06	0.015	4	4
SS30	2	0.125	0.25	0.015	8	8
SS34	1	0.125	0.125	0.015	8	8
SS36	1	0.25	0.125	0.06	8	4
SS37	≥8	0.125	0.5	0.03	≥16	4
SS38	1	0.125	0.125	0.03	8	4
SS39	1	0.125	0.125	0.008	8	16
SS43	>8	0.125	1	0.03	>8	4
SS53	1	0.25	0.125	0.06	8	4
SS73	1	0.125	0.06	0.03	17	8
SS76	≥8	0.5	0.5	0.125	≥16	4

^aFC, fold change; Comb, antibiotics combination.

FIG 3.

Interaction between AZM and ESC results in growth inhibition. The fractional inhibitory concentration (FIC) index for each strain treated with drug combinations is shown for AZM plus CFM (A) and AZM plus CRO (B). The red font indicates the specific FIC index. AZM, azithromycin; ESC, extended-spectrum cephalosporin; CFM, cefixime; CRO, ceftriaxone.

DISCUSSION

In this study, we evaluated the antimicrobial surveillance of 5,808 N. gonorrhoeae isolates during an 8-year period and clarified the status of drug resistance. In short, most isolates were resistant to PEN, CIP, and TET, which was consistent with findings in other regions, such as America, the Caribbean, and Southeast Asia (21, 22). SPT^r strains have been detected only sporadically over the past few years, indicating the absence of large-scale resistance transmission. Thus, once the ESC and AZM regimens have failed, SPT may represent a second option for further treatment, but it is still not suitable for pharyngeal gonorrhea (23). Moreover, the rates of AZM^r and ESC^d remained high and presented an upward trend. The number of isolates with CRO MIC of  ≥0.125 mg/liter increased considerably, and the proportion has reached 16.18%, which far exceeds that in other countries (24). Overall, most classic single-drug treatments might not be suitable as clinical medication regimens because of the high prevalence of antibiotic resistance.

Since 23S rRNA mutation (25–27) and penA mosaicism (28–30) were associated with AZM^r and ESC^d (31), we compared gene alleles and antibiotic MICs to explore the relationship between molecular features and antibiotic susceptibility. In this study, 23S rRNA was classified into wild-type 23S rRNA-100 and 23S rRNA-12 alleles and mutant 23S rRNA-1 and 23S rRNA-2 alleles, whose proportions were 87.34%, 1.27%, 6.33%, and 5.06%, respectively (see Table S1A in the supplemental material). Allele 1 and allele 2, featuring the A2059G and C2611T mutations, were highly associated with increased AZM resistance. Furthermore, 15 isolates shared mosaic penA alleles, including penA-10.001, penA-10.003, penA-34.007, penA-166.001, and the especially notable isolate penA-60.001 (Fig. 2B). Given the broad significance of penA-60.001, understanding its molecular features and evolution is a topic of critical importance. Finally, along with the MICs (Table S2), the molecular characteristics of 23S rRNA and penA could accurately reflect AZM^r and ESC^d. The occurrence and dissemination of resistant strains in Guangdong, China, has highlighted the necessity of molecular epidemiological surveys (32).

In response to the emergence of MDR N. gonorrhoeae strains, dual therapy was recommended by the WHO and is currently used as the first-line treatment in Canada (33) and Australia (34). From a clinical perspective, drug combination therapy is desirable because it allows lower concentrations of medicine to be used, which could treat infections with MDR clones and minimize the potential for side effects due to overdose. However, the role of dual therapy has been increasingly questioned in the context of AZM^r and ESC^d.

As reported, the CDC recommended a single 250-mg intramuscular (i.m.) dose of CRO and a single 1-g oral dose of AZM for the treatment of uncomplicated gonococcal infections as a strategy for preventing CRO resistance and treating possible coinfection with Chlamydia trachomatis in 2010 (35). However, in conjunction with the continued low incidence of decreased susceptibility to CRO (<1%) and the increased incidence of AZM resistance (from 0.6% in 2013 to 4.6% in 2018), the CDC updated the guidelines, recommending a single 500-mg i.m. dose of CRO for the treatment of uncomplicated gonorrhea (36). The dramatic rise in AZM resistance rates, especially among high-level tolerant strains, over the past few years has led European countries and the United States to reconsider the use of dual therapy in the treatment of gonorrhea (37). In China, CRO delivered as a single 1-g i.m. dose has been the main regimen for N. gonorrhoeae treatment. However, with the continuous increase in the numbers of high-level ESC-resistant strains (Fig. 1A, F, and G) and the side effects from high doses, the current therapy might not be suitable for further prevention and treatment of N. gonorrhoeae infection in Guangdong, China.

Because ESCs are the last remaining options for empirical gonorrhea monotherapy and in vitro susceptibility to CRO or CFM has slightly increased or stabilized since the introduction of dual therapy (38), with no clinical evidence to suggest that current dual-therapy effectiveness is waning, AZM plus ESC may be an effective treatment method before the application of new drugs in an environment of markedly increasing antibiotic resistance. However, to better verify the effectiveness of dual therapy, more high-level dual-resistant strains must be included in our follow-up research. Moreover, since AZM has a far longer half-life than ESC, it is not clear that a single dose of the two drugs given simultaneously will have any significant overlap regarding their area under the concentration-time curves (AUC), although, regarding dynamics, it has been confirmed that these two drug combinations showed synergistic effects regardless of the order of application (39). Due to the lack of animal experiments, we still cannot elucidate the mode of action and efficacy of dual therapy from an in vivo perspective.

In conclusion, we reported a high frequency of antibiotic resistance in Guangdong, China, which reveals that MDR gonorrhea is a major public health threat. Before the application of new antibiotics, dual therapy might be a recommended solution.

MATERIALS AND METHODS

Neisseria gonorrhoeae isolation and culture.

A total of 5,808 strains were collected from 2013 to 2020 in Guangdong Province, China, by the Gonococcal Antibiotic Resistance Monitoring Network operated by the Dermatology Hospital of Southern Medical University/Guangdong Provincial Dermatology Hospital. Additional details about the origins of the samples are provided in the supplemental material. Over time, we collected an increasing number of strains across almost all areas of Guangdong Province. The isolates were then identified by oxidase, catalase, and sugar fermentation tests, as recommended by the WHO. Finally, the isolates were cultured in Thayer-Martin medium and incubated in 5% CO₂ in a 37°C incubator.

The antibiotic susceptibility of N. gonorrhoeae isolates in 2016 was provided by Xingzhong Wu, and the decreased susceptible rate of CRO from 2016 to 2019 was published previously (data are used with the permission) (18, 40). The relevant published data are presented here to more accurately and objectively reflect the situation of antibiotic treatment in Guangdong, China.

Antimicrobial susceptibility testing.

Susceptibility to the antimicrobials PEN, CIP, TET, spectinomycin (SPT), AZM, CRO, and CFM was tested using the agar dilution method according to the WHO recommendations. Briefly, all the strains were cultured for 18 h and adjusted to a 0.5 McFarland standard suspension, and the cultures were then inoculated at multiple points on antimicrobial agar plates containing drugs at different concentrations. WHO D, G, J, L, K, and P were used as reference strains. Strains with MICs for PEN of ≥1.0 mg/liter, CIP of ≥1.0 mg/liter, Tet of ≥1.0 mg/liter, SPT of ≥128 mg/liter, and AZM of ≥1.0 mg/liter were classified as resistant, and those with MICs for CRO of ≥0.125 mg/liter and CFM of ≥0.25 mg/liter were identified as having decreased sensitivity (3).

Dual-therapy efficacy testing.

The antimicrobial susceptibility to AZM combined with CFM or CRO was tested. Briefly, all strains were cultured for 18 h and adjusted to a 1 McFarland standard suspension, and then 100 μL of the mixture was aliquoted into each well of a 96-well microplate containing different drug concentrations. WHO L and P were used as reference strains.

Sequence typing and phylogenetic analysis.

A total of 79 MDR isolates from Guangdong Province were suspended in 200 μL of 1× phosphate-buffered saline (PBS) and then heated in a boiling water bath for 10 min. The cell suspensions were centrifuged at 15,000 rpm for 5 min, and the supernatants were collected for further N. gonorrhoeae antimicrobial resistance (NG-STAR), N. gonorrhoeae multiantigen sequencing (NG-MAST), and multilocus sequence typing (MLST) analyses. Subsequently, PCR products were purified using a PCR purification kit from Sangon Biotech, and DNA sequencing was performed at Sangon Biotech Co., Ltd., using the Sanger sequencing method. Ultimately, the MLST genes (including aroE, adk, gdh, abcZ, fumC, pgm, and pdhC) were assembled and used for phylogenetic analysis. The analysis methods used to construct and test a neighbor-joining tree were maximum likelihood and bootstrap tests; MEGA7 was used to visualize the phylogenetic tree. The primers and annealing temperatures used for genotyping are shown in the supplemental material.

Synergy testing.

Dual therapy for ESC combined with AZM was investigated, and the effectiveness was determined by calculating the FIC index. The FIC was calculated by the following formula: $\text{FIC\hspace{0.17em}}=\left({\text{MIC}}_{\text{extended-spectrum\hspace{0.17em}cephalosporin}}^{\text{combination}}/{\text{MIC}}_{\text{extended-spectrum\hspace{0.17em}cephalosporin}}^{\text{alone}}\right)+\left({\text{MIC}}_{\text{azithromycin}}^{\text{combination}}/{\text{MIC}}_{\text{azithromycin}}^{\text{alone}}\right)$; an FIC of ≤0.5 indicates synergy.