Antimicrobial Susceptibility of Ertapenem in Neisseria gonorrhoeae Isolates Collected Within the China Gonococcal Resistance Surveillance Programme (China-GRSP) 2018

Introduction

Gonorrhea, caused by Neisseria gonorrhoeae, is one of the commonly seen sexually transmitted diseases with a total global incidence of approximately 86.9 million per year.¹ N. gonorrhoeae can cause urogenital infection and extragenital infection. It usually leads to dysuria and urethral discharge in men and frequently causes asymptomatic genital tract infection in women. If left untreated, it may lead to severe sequelae such as pelvic inflammatory disease and infertility.² Moreover, it can even facilitate the risk of HIV acquisition and transmission.³ To prevent complications and control the transmission, successful treatment is of great significance.

Ceftriaxone and azithromycin in various doses are recommended as first-line treatments in guidelines in America,⁴ Australia,⁵ Canada,⁶ Europe,⁷ New Zealand,⁸ and by the World Health Organization (WHO).⁹ Monotherapy with ceftriaxone (1 g intramuscularly in a single dose) for uncomplicated gonococcal infections is recommended as the first-line treatment according to the current guidelines in China.¹⁰ However, N. gonorrhoeae has developed resistance to all previously used antimicrobial therapies and shows decreased susceptibility to ceftriaxone (DSC, defined as isolates with ceftriaxone MIC ≥0.25 mg/L in China currently) which poses a significant global healthcare burden.^11–13 The emergence of N. gonorrhoeae isolates with a multidrug resistant or extremely drug-resistant profile highlights the importance of considering alternate therapy. Noteworthy, one treatment failure with 1 g ceftriaxone was reported in UK, but that infection was cleared with intravenous ertapenem 1 g once daily for 3 days.¹⁴

Ertapenem belongs to the class of carbapenem antibiotics, which interacts with penicillin binding proteins (PBPs) located on the bacterial cell wall. In particular, its inhibitory effect to PBPs 2 and 3 disturbs the final transpeptidation step in the synthesis of peptidoglycan, an essential component of the bacterial cell wall. Subsequently, dysfunctional cell wall causes cell death of Gram-positive and Gram-negative aerobic and anaerobic pathogens. According to the elimination half-life of ertapenem in healthy volunteers (3.8 hours), it is suitable for both intramuscular injection and intravenous.¹⁵ Besides its stability for DHP-1 (dehydropeptidase-1), one injection per day without combination of cilastatin is sufficient in clinical practice. Furthermore, there has been research proving that ertapenem can be regarded as a promising alternative for the treatment of ceftriaxone resistance both in vitro and in vivo. For instance, in the UK and Australia, ertapenem has been successfully applied to patients with multidrug resistant gonorrhea.^14,15 Additionally, in vitro studies have shown that isolates resistant to ceftriaxone, including isolates with high levels of resistance to ceftriaxone, were still sensitive to ertapenem.^16,17

Based on successful clinical treatment cases and data of previous in vitro studies,^16,17 in this study, we analyzed the susceptibility of ertapenem of 503 N. gonorrhoeae isolates. Those isolates were collected in 2018 from eight different Chinese provinces.

Materials and Methods

Gonococcal Isolates

The ethics approval for the China Gonococcal Resistance Surveillance Programme (China-GRSP) was obtained from the Medical Ethics Committee at the Institute of Dermatology, the Chinese Academy of Medical Sciences & Peking Union Medical College and the National Center for Sexually Transmitted Disease Control at Nanjing (2014-LS-026) for the use of specimens collected annually from patients attending local dermatology or STD clinics. Participants no less than 18 years of age who signed an informed consent to provide urine, vaginal and rectal swabs were enrolled in the study. The isolates detected in our study were randomly selected from the China-GRSP sample bank collected in 2018. Samples were inoculated, identified, preserved, and transferred as previously described.¹⁸

Clinical gonococcal isolates from eight provinces (Beijing, Guangdong, Guangxi, Liaoning, Sichuan, Shaanxi, Tianjin and Yunnan) were used. In total, there are 503 isolates, which were all collected from the urogenital tract. Among them, 415 isolates were selected out of random sampling for MIC distribution analysis in different provinces, and the other 88 isolates were additionally selected with preference (isolates with relatively high MICs of ceftriaxone or azithromycin) for cross resistance analysis.

Antimicrobial Susceptibility Tests by Agar Dilution

Antimicrobial susceptibility tests to ertapenem, ceftriaxone, and azithromycin were determined for all isolates by the agar dilution method as previously described.¹⁹ For ertapenem, the concentrations were 0.008, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, and 1 mg/L, for ceftriaxone they were 0.008, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, and 1 mg/L, and for azithromycin they were 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, and 4 mg/L. The lowest antibiotic concentration that inhibited growth was defined as the MIC of isolates. Resistance to azithromycin (RTA) was defined as MIC ≥ 1.0 mg/L, and DSC was defined as MIC ≥ 0.25 mg/L. So far, no standardized criteria for ertapenem to N. gonorrhoeae has been published from EUCAST and Clinical and Laboratory Standards Institute (CLSI). Quality assurance was taken using WHO N. gonorrhoeae reference isolates (G, J, K, L, and P) and ATCC 49226.²⁰

Data Analysis

The MIC of the reference strain was within the reference range whereas the batch of results was included in the analysis. 415 isolates from eight provinces were used to evaluate the MIC distribution in various province in China. Other analyses were carried out using all 503 isolates. Here, we describe the eight provinces’ MICs distribution of collected N. gonorrhoeae isolates to ertapenem. Correlation between MICs of ertapenem and ceftriaxone or azithromycin was calculated using log₂-transformed MIC data by linear regression among 503 isolates. For MICs detected as ≤ or ≥ a specific value, these specific values were used for the calculation of log₂-transformed MIC data. A p-value <0.05 was considered statistically significant. Statistical analyses were carried out utilizing SPSS software (IBM, New York, USA), and Excel (Microsoft, Washington, USA). Figures were made using R (GNU System).

Results

Antimicrobial Susceptibility Test of 415 Isolates

Geographically, the 415 isolates were collected from Beijing, Guangdong, Guangxi, Liaoning, Sichuan, Shaanxi, Tianjin, and Yunnan. The specific numbers of isolates for each province were 40, 59, 53, 38, 67, 52, 48, and 58, respectively.

All isolates were determined for their MIC values to ertapenem, ceftriaxone, and azithromycin. MIC values obtained from the WHO N. gonorrhoeae reference strains were identical or within 1 MIC dilution of those previously reported in every batch of tests.²⁰ It supported the reliability of the MIC values to those three antimicrobials.

Ertapenem MICs ranged from ≤0.008 to 0.5 mg/L. The MIC proportions of ertapenem in different provinces is shown in Figure 1. Strains (12 out of 415, 2.9%) with MIC greater than 0.25 mg/L were found in six provinces. Among them, the largest proportion (4 out of 12) were strains from Guangdong Province. Meanwhile, no strains with a MIC ≥ 0.25 mg/L were found in Liaoning and Tianjin (Table 1). In addition, there was one strain with MIC greater than 0.5 mg/L found in Shaanxi province (Table 1 and Figure 1). Overall, the MIC₅₀ of the eight provinces was 0.06 mg/L, with the exception of Sichuan and Yunnan provinces, those of which were both 0.03 mg/L (Table 1). While the overall MIC₉₀ of the eight provinces was 0.125 mg/L, although those of Liaoning, Shaanxi, Tianjin and Yunnan were 0.06 mg/L, the other four provinces were consistent with the overall value (Table 1). The difference between MIC₅₀ and MIC₉₀ values in different provinces suggested that the MIC distribution of ertapenem could be inconsistent among regions.

Table 1 The Proportions of the Isolates with Different MICs of Ertapenem by Provinces

Figure 1 The percentage of N. gonorrhoeae isolates with different MICs (mg/L) for ertapenem. The MIC50 (yellow-dotted line), MIC90 (red-dotted line) and number of isolates per province are shown. Abbreviations: BJ, Beijing; GD, Guangdong; GX, Guangxi; LN, Liaoning; SC, Sichuan; SX, Shaanxi; TJ, Tianjin; YN, Yunnan; n, number.

Correlation Between MICs of Ertapenem and Ceftriaxone or Azithromycin

MIC distribution and correlation analyses for ertapenem with ceftriaxone and azithromycin are shown in Figure 2. A correlation of r = 0.487 (P <0.001) was observed between ertapenem and ceftriaxone (Figure 2A). In comparison, the MIC values of ertapenem and azithromycin correlated poorly with r = −0.12 (P = 0.007) (Figure 2B). These results indicated a cross resistance between ertapenem and ceftriaxone. However, azithromycin resistance might have limited impact on ertapenem susceptibility.

Figure 2 Correlation between MICs of ertapenem and ceftriaxone or azithromycin. Each symbol represents one or multiple isolates, which is also shown with specific numbers and colors. The regression line was calculated using log2-transformed MIC data. For the MICs ≤ or ≥ a specific value, log2-transformed this specific value was used for calculating linear regression. (A) Correlation between MICs of ertapenem and ceftriaxone, r = 0.487, P <0.001. (B) Correlation between MICs of ertapenem and azithromycin, r = −0.12, P = 0.007.

MICs Distribution of Ertapenem in Isolates with Decreased Susceptibility to Ceftriaxone or Resistance to Azithromycin

Out of all 503 tested isolates, 24 isolates showed decreased susceptibility to ceftriaxone (DSC), 85 isolates were resistant to azithromycin (RTA), three isolates possessed a resistance to both ceftriaxone and azithromycin. Ertapenem MICs distributions for DSC or RTA isolates are shown in Table 2. Among the 24 DSC isolates, only four isolates showed a MIC ≥ 0.25 mg/L in ertapenem. Regarding RTA isolates, only three out of 85 showed MIC ≥ 0.25 mg/L for ertapenem.

Table 2 Ertapenem MICs Distributions for Decreased Susceptibility to Ceftriaxone (DSC) or Azithromycin-Resistant (RTA) N. gonorrhoeae Isolates

Discussion

The antimicrobial resistance of N. gonorrhoeae remains a severe issue for public health. Ceftriaxone-resistant isolates have been reported in many countries over the world,^{2,3,12,13,21,22} which suggests the importance of finding alternative treatments. The recommended strategies from the World Health Organization include “increasing the therapeutic dose”, “new antimicrobial drugs development”, “recycle “old” antimicrobial agents”, etc. The gonorrhea treatment guidelines in China suggest an increase of ceftriaxone dose in the treatment of gonorrhea (from 250 mg in 2014 to 1 g in 2020). However, this can only temporarily alleviate the dilemma of gonorrhea treatment. With the evolution of gonococcal drug resistance, highly ceftriaxone-resistant isolates may continue to emerge. Therefore, it is urgent to find other effective therapeutic drugs.

For the time being, new antibiotics are also under intensive development, such as zoliflodacin and gepotidacin.^23,24 However, the development of new drugs is known to be extremely time and cost-consuming. It is estimated that the research and development of new antimicrobials requires high socio-economic costs from target to product, which may take 15 years and $2.5 billion.²⁵ Compared with the drug R & D returns of other non-infectious and chronic diseases, the R & D of antimicrobials development has more market risks. Therefore, the reassessment of developed drugs is of great significance. It is not only one of the alternative treatment strategies recommended by the WHO, but also the fastest treatment for gonococcal resistance.

Studies have shown that ertapenem may be a good alternative to ceftriaxone-resistant isolates both in vitro and in vivo. In vitro studies showed that ertapenem was still sensitive to ceftriaxone-resistant isolates (including highly ceftriaxone-resistant isolates).¹⁶ In addition, there are reports about ertapenem successfully curing multidrug-resistant gonorrhea patients.¹⁵ However, N. gonorrhoeae can easily evolve antimicrobial resistant variants. Therefore, we suggest a parallel antimicrobial resistance surveillance of ertapenem if it is used in a larger scale.

In this study, we found that ertapenem demonstrated a low MIC value (MIC range: ≤0.008–0.5 mg/L) in isolates collected from eight provinces in China. Using the PK-PD (non-species related) breakpoints reported in EUCAST clinical breakpoint of ertapenem (MIC ≤ 0.5 mg/L, susceptible), all isolates were sensitive to ertapenem, which indicated that ertapenem might be a potential therapeutic agent in gonorrhea treatment. Moreover, the MIC₉₀ of ertapenem (0.125 mg/L) equaled the value of ceftriaxone in our study, but higher than that (MIC₉₀ 0.06 mg/L) reported by Yang et al., who had detected that level with 504 isolates collected from Zhejiang province from 2011–2012 and 2015–2017.²⁶ This variation might be related to the time periods of the selected isolates, or the differences between provinces, as we found that the MIC₉₀ in different provinces ranges from 0.06 to 0.125 mg/L in our study revealed the existence of variations in different provinces. The proportions of MIC ≥ 0.25 mg/L ranged from 0 (Liaoning and Tianjin) to 6.8% (Guangdong). Although this difference was not statistically significant, it could indicate the importance of antimicrobial susceptibility surveillance. Especially before prescribing antibiotics to patients, an antimicrobial sensitivity test is vitally important.

Since both ertapenem and ceftriaxone are located to β-Lactams, we analyzed the correlation between ertapenem and ceftriaxone MIC to evaluate the cross resistance. The MICs of ertapenem and ceftriaxone showed a correlation of r = 0.487, which indicated a cross resistance between ertapenem and ceftriaxone. But the MIC ranges of ertapenem in DSC isolates were lower than that of ceftriaxone, and in 24 DSC isolates, only four isolates showed ertapenem MIC ≥ 0.25 mg/L. Two of the 503 isolates had a MIC value of 0.5 mg/L. Whether these isolates will evolve into ertapenem-resistant isolates in the future will need further monitoring. Accordingly, ertapenem had in vitro advantages over ceftriaxone for isolates with ceftriaxone resistance. Generally clinicians are cautious about the use of carbapenem antibiotics, as they are usually used for serious infections in clinical practice. But facing high dose ceftriaxone treatment failure or persistent gonococcal infection patients, the clinicians may draw their attention to the application of ertapenem.

Our in vitro results suggested that ertapenem might be an effective treatment option for gonorrhea, particularly for the DSC isolates currently identified, which could serve as a foundation for further clinical trials in vivo. This study is the first multi-province research reporting the N. gonorrhoeae susceptibility to ertapenem in China. Moreover, our results provide a reference for antimicrobial susceptibility data of ertapenem in vitro for gonococcal infections in China. Nevertheless, there are some limitations in this study: the number of isolates accounts for only 0.3% (415/133,156) of the reported cases of gonorrhea in China for 2018, and lacking extragenital isolates in this study may also lead to a potential bias. At the same time, when interpreting the susceptibility data, we refer to the susceptibility break points of PK-PD (non-species related) reported in EUCAST, and there is no break point of antimicrobial susceptibility of ertapenem to N. gonorrhoeae, which may also lead to bias in the analysis of results. All these deficiencies may limit the generalizability of this study in China.

Conclusion

The study evaluated ertapenem MICs of N. gonorrhoeae isolates in 2018 from eight provinces in China and the correlation was analyzed to see the potential cross resistance between ertapenem and ceftriaxone or azithromycin. The antimicrobial effect of ertapenem on isolates with DSC and resistant to azithromycin were also evaluated. Our results suggest that ertapenem has low MIC values with isolates collected from eight provinces in China. Overall, this study provides new scope for ertapenem in the treatment of gonorrhea, although its effectiveness in vivo needs further study.