Neisseria gonorrhoeae Strain with Reduced Susceptibilities to Extended-Spectrum Cephalosporins

The spread of Neisseria gonorrhoeae strains with reduced susceptibility to extended-spectrum cephalosporins is an increasing public health threat. Using Etest and multiantigen sequence typing, we detected sequence type 1407, which is associated with reduced susceptibilities to extended-spectrum cephalosporins, in 4 major populated regions in California, USA, in 2012.

N eisseria gonorrhoeae infections are the second most common sexually transmitted infection in the United States (1). Gonorrhea typically presents as urethritis in men and cervicitis in women but when left untreated can result in severe sequelae, such as infertility (2,3). Gonorrhea infections continue to be a public health problem worldwide, and control efforts have been complicated because of the ability of the organism to develop resistance to all first-line antimicrobial drugs used in treatment, including penicillins, tetracyclines, and fluoroquinolones.
Treatment failures and isolates with reduced susceptibilities to extended-spectrum cephalosporins (ESCs) have been detected in Asia (4,5), Canada (6), Europe (7,8), and South Africa (9). In the United States, ESCs are currently the foundation of treatment recommendations. However, the increasing prevalence of isolates with reduced susceptibility to ESCs has led to dual treatment with ceftriaxone plus azithromycin or doxycycline. This combination is the only recommended treatment regimen of the US Centers for Disease Control and Prevention (CDC) (10).
N. gonorrhoeae isolates with reduced susceptibility to ESCs have been linked to altered penicillin-binding protein, which is encoded by the penA gene. Molecular and genomic epidemiologic studies have been used to describe N. gonorrhoeae antimicrobial drug resistance to ESCs in much of the world. In the United States, isolates with reduced susceptibility to cefixime have been associated with a specific strain of N. gonorrhoeae (11). However, little is known about the molecular epidemiology of strains with reduced susceptibilities to ceftriaxone, the current recommended ESC. In this study, we conducted surveillance for genotypic and phenotypic diversity of N. gonorrhoeae strain diversity in California during January 2012-September 2013.

The Study
Urethral gonococcal isolates were obtained from men with urethral infections during the CDC-sponsored Gonococcal Isolate Surveillance Project (GISP) at sites in Los Angeles, Orange, San Diego and San Francisco Counties in California. Duplicates of all GISP isolates from these sites were analyzed at the San Francisco Department of Public Health Laboratory by using Etest and at the GISP regional laboratory by using agar dilution. Antimicrobial drug susceptibility testing was performed by using Etest (bioMérieux, Marcy l'Etoile, France) as part of the California rapid response project to enhance surveillance efforts aimed at detecting treatment failures and containing the spread of isolates that require increased MICs of third-generation cephalosporins.
A total of 718 isolates were tested for susceptibility to ceftriaxone, cefixime, and azithromycin by Etest. In brief, frozen isolates were thawed in a biosafety cabinet at room temperature, inoculated onto a chocolate II agar plate containing 1% Iso VitaleX (Becton Dickinson, Franklin Lakes, NJ, USA), and incubated at 37°C with in an atmosphere of 5% CO 2 for 20-25 h. A sterile swab was then used to collect colonies on the plate and suspend them in typticase soy broth containing 15% glycerol to give a turbidity of 0.5-1 MacFarland units. Using another sterile swab, we plated out the liquid culture onto 3 chocolate II agar plates in three 90° turnings. We then placed an Etest strip in the center of each plate by using a manual applicator. After incubation for 20-25 h at 37°C in an atmosphere of 5% CO 2 , the plates were examined, and MICs of each drug were recorded according to the manufacturer's instructions.
Molecular epidemiologic typing was performed by using N. gonorrhoeae multiantigen sequence typing (NG-MAST) on all isolates. NG-MAST was performed as described (12) and allele numbers of porB and tbpB genes and sequence types (STs) were assigned by using the NG-MAST website (http://www.ng-mast.net).
Of 718 urethral isolates that were sequence typed, 258 STs were identified that had 192 porB alleles and 60 tbpB alleles. Eighty-three STs were present among ≥2 isolates in the collection, and the remaining 175 STs were unique. The 5 most prevalent STs identified were ST2400 (55, 7.7%), ST2992 (46, 6.4%), ST3307 (45, 6.3%), ST1407 (38, 5.3%), and ST7268 25, 3.5%). These STs constituted 29% of all isolates obtained. ST1407, the fourth most prevalent ST in this study, has been associated with ESC treatment failures in Europe and Canada. In California, this ST and closely related STs were associated with reduced susceptibility to ESCs, but these STs remained susceptible to azithromycin.
Most isolates in our study required low MICs (≤0.023 µg/mL) for ceftriaxone and cefixime. Proportions of low, intermediate, and high MICs required by isolates in the 5 most prevalent genogroups are shown in Table 2.
Seventeen isolates that had reduced susceptibility phenotypes for ESCs (ceftriaxone MIC ≥0.125 µg/mL and cefixime MIC ≥0.125 µg/mL) were subjected to penA sequencing to determine the presence an altered penA gene. Sequencing was performed as described (14). Sequencing of penA genes showed that 100% (17/17) had the mosaic penA XXXIV allele. Genetic characteristics of isolates that required high MICs of ESCs are shown in Table 3. The tbpB 110 allele was found in 100% (17/17) of the isolates that required high MICs of ESCs Cs, and 94% (16/17) were G1407.

Conclusions
Genotypic surveillance of N. gonorrhoeae has been performed in the United States and elsewhere, but little is known about the genetic diversity of strains circulating  in the United States. We sought to perform phenotypic and genotypic surveillance of isolates from major population centers in California in 2012. ST1407, a widespread strain linked to ESC treatment failures, was found in all 4 counties in California and was the fourth most prevalent ST overall. In this study, ST1407 was associated with reduced susceptibility to ceftriaxone and cefixime. These data show that ST1407 is well established in California and are consistent with results from Asia (4,5), Canada (15), Europe (8), and the United States (11). All G1407 isolates that required increased MICs of ceftriaxone or cefixime had penA allele XXXIV. Among the 718 isolates in this study, none had reduced susceptibility to azithromycin and an ESC. The relatively high prevalence of this strain type, combined with the evolutionary capacity of N. gonorrhoeae to evade antimicrobial drug pressure, may eventually lead to circumstances where even dual therapy is not sufficient.