Association of Neisseria gonorrhoeae genogroups and specific PBP2/MtrR/PorB mutation patterns with susceptibility to penicillin in a susceptible gonococcal population.

Objectives: To ascertain whether the antimicrobial susceptibility of Neisseria gonorrhoeae isolates with differing susceptibilities to penicillin is associated with genogroups (GGs) and combined mutation patterns in PBP2 (penA), the multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB). Methods: The susceptibility of 146 clinical N. gonorrhoeae isolates to penicillin was determined using the agar dilution method and the interpretation criteria of CLSI. The DNA sequences of penA, mtrR and porB in isolates were compared with WT sequences and mutation patterns were determined. Isolates were typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and STs were grouped into specific GGs. Results: The isolates tested carried 9 mutation patterns in PBP2 and 12 mutation patterns in each of MtrR and PorB. Of the 146 isolates, 121 (82.9%) were grouped into 13 different GGs. Isolates with penicillin MICs of 0.03-0.06 mg/L were significantly associated with GG25 (P < 0.05) and PBP2/MtrR/PorB mutation pattern I/WT/WT (P < 0.01). Isolates with a penicillin MIC of 1.0 mg/L were associated (P < 0.05) with: (i) GG3655 and mutation pattern XXII/A-;G45D/G120K;A121N; (ii) GG921 and mutation pattern IX/G45D/G120D;A121N; and (iii) GG1109 and mutation pattern IX/G45D/WT. Sixty percent (9/15) of penicillin-resistant isolates (MIC ≥2 mg/L) were GG3654 (P < 0.0001) and carried mutation pattern IX/G45D/G120K;A121D or IX/G45D/G120D;A121D (P < 0.05). Conclusions: Specific mutation patterns in PBP2/MtrR/PorB were associated with specific GGs and penicillin susceptibility. This approach of typing strains and resistance patterns is ideal for predicting antimicrobial resistance and should be used in instances in which gonococcal culture is not available but DNA can be obtained from clinical specimens.

Suitability of a chocolate agar-based medium for the transportation and long term storage of Neisseria gonorrhoeae isolates at room temperature.

Neisseria gonorrhoeae isolates were grown on chocolate agar slants comprising GC Agar Medium Base supplemented with 1% modified Kellogg's supplement and 1% beef blood hemoglobin. The bacterial growth was covered with sterilized paraffin oil. Cultures were used for transportation and storage of Neisseria gonorrhoeae for 3months at room temperature.

High levels of susceptibility to new and older antibiotics in Neisseria gonorrhoeae isolates from Saskatchewan (2003-15): time to consider point-of-care or molecular testing for precision treatment?

OBJECTIVES: The antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Saskatchewan was determined retrospectively (2003-15) to ascertain temporal trends to both current and older antimicrobials used for treatment. METHOD: The agar dilution method was used to test the antimicrobial susceptibilities of 685 isolates to seven antibiotics. RESULTS: Over the period, only three (0.4%) gonococcal isolates had reduced susceptibility to cefixime and/or ceftriaxone. All isolates were susceptible to spectinomycin. Over 95% of the isolates tested were susceptible to azithromycin except in 2010 and 2013 (27.6% and 7.2% resistant, respectively). One isolate was resistant to both azithromycin and cefixime. Ciprofloxacin resistance was seen in < 5% of isolates prior to 2010, but in > 5% thereafter. From 2006 to 2012, and in 2015, penicillin resistance was detected in < 5% (0%-4.0%) of isolates, but in > 5% for the rest of the study period. Tetracycline resistance remained >5% (11.8%-89.1%) throughout the study. Plasmid-mediated resistance to tetracycline fluctuated between 0% and 17.5% of isolates tested. Four isolates were MDR and two isolates were XDR. CONCLUSIONS: N. gonorrhoeae isolates were largely susceptible (∼85%) to antibiotics no longer recommended for treatment, such as penicillin and ciprofloxacin. Gonorrhoea in Saskatchewan is primarily (>95%) diagnosed by nucleic acid amplification testing, which does not permit antimicrobial susceptibility testing. The development of molecular testing, or point-of-care tests, to evaluate antimicrobial susceptibility, would enhance knowledge of true levels of resistance and allow discretion as to whether older but still effective antibiotics could be used in individual patient care.

Using crude whole-genome assemblies of Neisseria gonorrhoeae as a platform for strain analysis: clonal spread of gonorrhea infection in Saskatchewan, Canada.

Using crude whole-genome assemblies, we analyzed 25 isolates of Neisseria gonorrhoeae by using a high-resolution single nucleotide polymorphism (SNP) approach for nine housekeeping genes, characterizing penA alleles, and antimicrobial susceptibility phenotypes coupled with population structure analysis. Two clonal complexes, characterized by their spatial and geographical persistence, were identified. In addition, the clonal spread of penicillin-resistant/intermediate phenotypes and a novel introduction of the azithromycin resistance phenotype in Saskatchewan, Canada, were ascertained using this method.

Molecular epidemiology of Neisseria gonorrhoeae isolates from Saskatchewan, Canada: utility of NG-MAST in predicting antimicrobial susceptibility regionally.

OBJECTIVES: To investigate the molecular epidemiology of isolates of Neisseria gonorrhoeae from Saskatchewan, Canada, using Neisseria gonorrhoeae multi antigen sequence typing (NG-MAST), and to assess associations between antimicrobial susceptibility (AMS) and specific strain types (STs). METHODS: 320 consecutive gonococcal isolates, collected between 2003 and 2008, were typed by NG-MAST. STs were grouped if one of their alleles was common and the other differed by ≤1% in DNA sequence. AMS was determined by agar dilution (CLSI) to seven antibiotics. RESULTS: N gonorrhoeae isolates were resolved into 82 individual NG-MAST STs and 18 NG-MAST ST groups with groups 25, 3655, 921, 3654, 3657 and 3656 comprising 53.4% (171/320) of the isolates. N gonorrhoeae isolates susceptible to all the tested antimicrobials were significantly (p<0.05) associated with ST 25 (87%). Other significant associations between ST and AMS included: ST 3654 and isolates with minimum inhibitory concentrations of ≥0.03 mg/L to third generation cephalosporins; ST 3711 (100%) and TRNG; and ST/group 3654 (43%) and chromosomal resistance to penicillin and tetracycline. Several NG-MAST STs/groups were significantly associated with isolates with chromosomal resistance to tetracycline. Isolates resistant to ciprofloxacin (n=5) and azithromycin (n=2) appeared as individual STs. Significant associations were observed among individual STs, sex and age of the patient, and regional and temporal distributions. CONCLUSIONS: Associations between N gonorrhoeae AMS and NG-MAST STs were identified and may be useful in predicting AMS regionally. Because STs in different countries vary considerably, the use of NG-MAST for the prediction of AMS globally requires further study.

Longitudinal analysis of the evolution and dissemination of Neisseria gonorrhoeae strains (Saskatchewan, Canada, 2005 to 2008) reveals three major circulating strains and convergent evolution of ciprofloxacin and azithromycin resistance.

A longitudinal study combining multilocus sequence typing with molecular evolutionary analysis determined the distribution, population structure, and evolution of antibiotic resistance in Neisseria gonorrhoeae isolates in Saskatchewan that were collected between 2005 and 2008. Of 195 gonococcal isolates examined, 29 sequence types (STs) were identified with 3 major circulating strains (ST-1 through ST-3) comprising 52% of all gonococcal isolates studied. The prevalences, persistence, distribution patterns, and clonalities of these isolates strongly suggest that gonorrhea endemicity within this broad geographic region was driven by these 3 circulating strains. ST-1 exhibited a significantly (P = 0.001) higher prevalence throughout the study than did the others, accounting for ∼25% of the tested isolates each year. The spatial distributions of the gonococcal strains indicated that ST-1 in 2007 entered a linear component of the sexual network, reaching the remote north and resulting in the further spread and maintenance of infection. Ciprofloxacin and azithromycin resistances were observed in distantly related gonococcal lineages, clearly indicating the convergent acquisition of these antibiotic-resistant phenotypes. In addition, all ciprofloxacin- and azithromycin-resistant lineages were found at the edges of the minimum spanning tree, far from the major lineages, suggesting that these antibiotic phenotypes were most likely introduced into the province. In contrast, resistance to penicillin was found mostly in the endemic gonococcal lineages, suggesting that penicillin resistance was probably acquired in Saskatchewan as a result of spontaneous mutations in already-established lineages. Tetracycline resistance was present in all STs except one, indicating its ubiquitous nature in the gonococcal population studied.

Extracts of Canadian first nations medicinal plants, used as natural products, inhibit neisseria gonorrhoeae isolates with different antibiotic resistance profiles.

BACKGROUND: Neisseria gonorrhoeae (Ng) has developed resistance to most antimicrobial agents and the antibiotics recommended for therapy are restricted, for the most part, to third generation cephalosporins. In order to investigate new potential sources of antimicrobial agents, the antibacterial properties of 14 Canadian plants used in traditional First Nations' medicine were tested against Ng isolates having differing antimicrobial susceptibility profiles. METHODS: Ethanolic extracts of 14 Canadian botanicals, analyzed by high-performance liquid chromatography, were tested for their antimicrobial activity (disc diffusion and/or agar dilution assays) against susceptible Ng reference strains and a panel of 28 Ng isolates with various antimicrobial resistance profiles. RESULTS: Extracts of Arctostaphylos uva ursi (kinnikinnick or bearberry), Hydrastis canadensis (goldenseal), Prunus serotina (black cherry), and Rhodiola rosea (roseroot) inhibited the growth of all Ng isolates with minimum inhibitory concentrations of 32 µg/mL, 4 to 32 µg/mL, 16 to >32 µg/mL, and 32 to 64 µg/mL, respectively. Extracts of Acorus americanus (sweet flag), Berberis vulgaris (barberry), Cimicifuga racemosa (black cohosh), Equisetum arvense (field horsetail), Gaultheria procumbens (wintergreen), Ledum groenlandicum (Labrador tea), Ledum palustre (marsh Labrador tea), Oenothera biennis (common evening primrose), Sambucus nigra (elderberry), and Zanthoxylum americanum (prickly ash) had weak or no antimicrobial activity against the Ng isolates with minimum inhibitory concentrations ≥256 µg/mL. The phytochemical berberine from H. canadensis inhibited the growth of all Ng isolates. The phytochemicals, salidroside and rosavin, present in R. rosea, also showed inhibitory activity against Ng strains. CONCLUSION: Canadian botanicals represent a potential source of novel compounds which inhibit Ng, including isolates resistant to antibiotics.