Molecular mechanisms of antimicrobial resistance and population dynamics of Neisseria gonorrhoeae in Saskatchewan (2003-2011)

Abstract

Gonorrhea is caused by the human pathogen Neisseria gonorrhoeae. More than 106 million new cases of N. gonorrhoeae infections occur each year worldwide. There is no vaccine available against gonococcal infections and treatment of gonorrhea with antibiotics is the only way to eradicate infection. The high prevalence of antibiotic resistance (AMR) in this microorganism makes the effective treatment of gonococcal infections increasingly problematic. The emergence of AMR, especially to extended spectrum cephalosporins (i.e. cefixime and ceftriaxone) which are the last possibilities for single dose treatment options for gonococcal infections, is a serious concern. Gonorrhea may become an untreatable infection in the near future.

Saskatchewan (SK) has one of the highest rates of gonorrhea in Canada. In order to better characterize the gonorrhea epidemic in SK, the objectives of the present research were to determine the prevalence and trends of AMR and emerging AMR mechanisms in N. gonorrhoeae isolates. AMR mechanisms were ascertained for the first time in SK in order to identify genetic causes of resistance. This was completed by determining and analyzing the DNA sequences of various genes - penA, mtrR, porB ponA, gyrA, parC mtrR, 23S rRNA alleles and erm –implicated in gonococcal AMR. The population dynamics of the N. gonorrhoeae isolates in SK was investigated by DNA based molecular methods to determine strain distribution, evolution of AMR phenotypes, and association between strain types (STs) and AMR genotypes and phenotypes.

N. gonorrhoeae isolates (n=427) from Saskatchewan (2003-2011) were susceptible to antibiotics now recommended for treatment - cefixime, ceftriaxone and spectinomycin. Over 95% of the isolates tested were also susceptible to penicillin (96%) and ciprofloxacin (95.5%), antibiotics no longer recommended for treatment, and azithromycin (99.4%). Tetracycline resistance was also high (50.1%).

N. gonorrhoeae isolates that were resistant to the antibiotics tested and also those isolates with MICs ≥0.003 mg/L to cefixime and ceftriaxone were analyzed (n=146) to determine their resistance mechanisms. This analysis revealed that reduced susceptibility to ceftriaxone and cefixime and resistance to penicillin is mediated by specific mutations in penicillin binding protein 2 (PBP2), in the promoter and dimerization domains of MtrR and porin protein (PorB). Novel mutations and combinations of mutations were noted. Ciprofloxacin resistant N. gonorrhoeae isolates carried double mutations in GyrA (S91F and D95G/N) and a S87R or S88P substitution in ParC. Isolates resistant to azithromycin had specific mutations in all the four alleles of 23S rRNA as well as in the DNA binding domain of MtrR. Most resistance was chromosomally mediated while plasmid-mediated resistance to penicillin (0.93% of penicillin resistant isolates) and tetracycline (3.3%) was low.

DNA based strain typing methods such as porB-DNA sequencing, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) showed that the gonococcal population in SK differs appreciably from both other Canadian provinces and from strains reported internationally. MLST analysis, which ascertains the evolution of isolates over time, demonstrated that penicillin and tetracycline resistant isolates in SK evolved through spontaneous mutations in established lineages. Ciprofloxacin and azithromycin resistant N. gonorrhoeae isolates, on the other hand, were introduced into SK from outside the province. Significant associations between particular mutation pattern combinations in resistance determining genes and specific NG-MAST STs were identified e.g. NG-MAST ST 25 was associated with specific combined mutation patterns in PBP2, MtrR and PorB and antibiotic susceptibility; and, NG-MAST ST 3654 was associated with another PBP2/MtrR/PorB mutation pattern, chromosomal resistance to penicillin and tetracycline and elevated MICs to cefixime.

This research shows the importance of regional antimicrobial susceptibility monitoring. In the context of SK, this means that local surveillance of gonococcal AMR may be used to develop policies for regional treatment guidelines which promote the prudent use of antimicrobials for treatment, including those antibiotics which may no longer be used in other regions due to higher AMR rates. Further, the significant association between particular AMR mutation pattern combinations and specific STs indicates that AMR might be predicted. These results should assist in the development of non-culture-based tests for the diagnosis of gonococcal AMR similar to nucleic acid amplification tests used to diagnose N. gonorrhoeae infections.