Sidharath D. Thakur

Using Crude Whole-Genome Assemblies of Neisseria gonorrhoeae as a Platform for Strain Analysis: Clonal Spread of Gonorrhea Infection in Saskatchewan, Canada

ABSTRACT 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

ABSTRACT 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.

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.

Resistance to Ceftriaxone and Azithromycin in : 2010–2011 neisseria gonorrhoeae : 2010–2011 Isolates From 7 Countries of South America and the Caribbean: 2010–2011

Seven countries in Latin America and the Caribbean report on (2010 and 2011) the susceptibility of 2235 isolates of Neisseria gonorrhoeae to 6 antibiotics. Thirteen isolates had ceftriaxone minimum inhibitory concentrations (MICs) of 0.125 to ≥ 0.25 mg/L. The percentage of resistant isolates to the following antibiotics was: azithromycin, 1.0% to 1.7%; ciprofloxacin, 42.1% to 36.2%; penicillin, 31% to 35%; tetracycline, 21.8% to 22.6%.

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 Kelloggs 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.

P3.289 MIC Creep to Ceftriaxone and Low Levels of Resistance to Azithromycin in 7 Countries from South America and the Caribbean

Background The World Health Organization (WHO) issued an international action plan in 2012 to mitigate the health impact of antimicrobial resistant Neisseria gonorrhoeae isolates. A key strategy is to strengthen international surveillance of gonococcal antimicrobial susceptibility. The Gonococcal Antimicrobial Surveillance Program (GASP) in Latin America and Caribbean (LAC) has reported on AMR trends from 1990. The present study presents regional trends in antimicrobial susceptibility between 2010 and 2011. Methods Seven countries reported using either agar dilution (CLSI), Etest or disc diffusion assays to determine antimicrobial susceptibility. Countries were asked to report MIC data and categories of susceptibility. Results Seven countries tested 1019 isolates of N. gonorrhoeae in 2010 and 1216 isolates in 2011 to ceftriaxone, penicillin, tetracycline and ciprofloxacin (n = 7); azithromycin (n = 4) and spectinomycin (n = 3). Several countries reported a 2-fold increase in MIC50 to ceftriaxone (from 0.004 to 0.008 µg/mL) between 2010 and 2011 and 12 isolates with ceftriaxone MICs 0.125- ≥ 0.25 µg/ml were reported in 2011. All isolates were susceptible to spectinomycin. Resistance to azithromycin increased slightly from 1. 0% (6/612) to 1.7% (20/1169) while resistance to ciprofloxacin decreased from 42.1% (429/1019) to 36.2% (439/1214) of isolates tested between 2010 and 2011. Resistance to penicillin increased from 31% (310/1016) in 2010 to 35% (428/1216) in 2011 while the percentage of isolates resistant to tetracycline was stable (2010 – 21.8%, 187/858; 2011 – 22.6%, 275/1216). Conclusions Third generation cephalosporins and spectinomycin continue to be viable options for the treatment of gonorrhoea in the countries reporting. Low percentages of resistance to azithromycin continue to be reported. There has been a steady decline in capacity for N. gonorrhoeae diagnosis and antimicrobial susceptibility testing in the region. The implementation of the WHO action plan to control the spread and impact of antimicrobial resistance in N. gonorrhoeae is an urgent priority.

Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Latin American and Caribbean countries, 2013–2015

Objectives A Neisseria gonorrhoeae antimicrobial susceptibility quality control comparison programme was re-established in Latin America and the Caribbean to ensure antimicrobial susceptibility data produced from the region are comparable nationally and internationally. Methods Three panels, consisting of N. gonorrhoeae isolates comprising reference strains and other characterised isolates were sent to 11 participating laboratories between 2013 and 2015. Antimicrobial susceptibilities for these isolates were determined using agar dilution, Etest or disc diffusion methods. Modal minimum inhibitory concentrations (MICs) for each panel isolate/antibiotic combination were calculated. The guidelines of the Clinical and Laboratory Standards Institute were used for interpretations of antimicrobial susceptibility. The agreement of MICs with the modal MICs was determined for each of the participating laboratories as well as for each of the antibiotics tested. Results Five of 11 laboratories that participated in at least one panel had an overall average agreement between participants’ MIC results and modal MICs of >90%. For other laboratories, agreements ranged from 60.0% to 82.4%. The proportion of agreement between interpretations for all the antibiotics, except penicillin and tetracycline, was >90%. The percentages of agreement between MIC results and their modes for erythromycin, spectinomycin, cefixime and azithromycin were >90%. Tetracycline, ceftriaxone and ciprofloxacin agreement ranged from 84.5% to 89.1%, while penicillin had 78.8% agreement between MICs and modal MICs. Conclusions The participating laboratories had acceptable results, similar to other international quality assurance programmes. It is important to ensure continuation of the International Gonococcal Antimicrobial Susceptibility Quality Control Comparison Programme to ensure that participants can identify and correct any problems in antimicrobial susceptibility testing for N. gonorrhoeae as they arise and continue to generate reproducible and reliable data.

P3.221 High levels of susceptibility in neisseria gonorrhoeae isolates from saskatchewan canada (2003–2015) to new and older antibiotics used for treatment

Introduction Antibiotics should have an efficacy of at least 95% for treating infections caused by Neisseria gonorrhoeae (Ng). When more than 5% of Ng isolates are resistant to an antibiotic, treatment guidelines must be changed to a more effective antibiotic. Trends in the antimicrobial susceptibility (AMS) of Ng isolates from Saskatchewan, Canada were ascertained (2003 – 2015) to ascertain whether older antimicrobials might be effective. Methods The susceptibility of 685 Ng isolates to 7 antibiotics was determined by the agar dilution method. β-lactamase production was determined using nitrocefin. Results From 2006–2012, penicillin resistance was below 5% (0%–4.0%) of Ng isolates tested. Penicillin resistance above 5% occurred in 2003 (6.7%), 2004 (6.8%), 2005 (11.5%), 2013 (27.5%) and 2014 (13.5%). Tetracycline resistance remained above 5% (11.8% to 89.1%) of Ng tested throughout the study. Ciprofloxacin resistance ranged between 0% and 1.9% of isolates tested up to 2009 but was over 5% thereafter. All isolates were susceptible to spectinomycin. Over 95% of Ng isolates tested were susceptible to azithromycin except in 2010 (27.6% resistant; 8/29) and 2013 (7.2% resistant; 5/69). Twelve (1.8%) isolates over the period showed reduced susceptibility to cefixime (2006 - 1/55; 2012 - 2/50; 2013 - 4/69; 2014 - 2/89; 2015 - 1/63) and/or ceftriaxone (2012 - 2/50). One isolate was resistant to both azithromycin and cefixime. Conclusions Cases of gonorrhoea in Saskatchewan (>95%) are diagnosed by nucleic acid testing with no AMS testing. Our research showed that many no-longer recommended antibiotics (penicillin, ciprofloxacin) were still effective over many years. Even with the recent higher percentages of Ng isolates resistant to penicillin and ciprofloxacin, ~87% of Ng in the province remains susceptible to these antibiotics. The development of NAATs to test for AMS 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.

P3.269 Association of Neisseria Gonorrhoeae NG-MAST Strain Types and Specific Mutation Pattern Combinations in penA, mtrR and porB

Background Antimicrobial resistance to third generation cephalosporins, penicillin and tetracycline in Neisseria gonorrhoeae isolates can be associated with particular strain types (STs) as well as specific mutation patterns in penA, mtrR or porB. With a view to developing molecular diagnostics for antimicrobial susceptibility, we investigated whether antibiotic resistant and susceptible N. gonorrhoeae isolates from Saskatchewan Canada were associated with specific STs and combined mutation patterns in penA, mtrR or porB. Methods DNA sequences of penA, mtrR and porB for 146 N. gonorrhoeae isolates were compared to “wild type” penA (GenBank#M32091), mtrR (GenBank#Z25796) and porB (GenBank#M21289) sequences. Mutation pattern numbers for penA were assigned as described by others. STs were ascertained by NG-MAST. Isolates were selected based on antimicrobial susceptibility phenotypes to 7 antibiotics. Results Strains were classified into 51 NG-MAST STs; 6 STs (86/146; 59%) comprised ≥ 5 isolates, 10 STs included 2–4 isolates, and 35 STs contained 1 isolate. Isolates with ST 25 (33/36, 92%) were associated (P < 0.0001) with penA/mtrR/porB pattern I/WT/WT and with antibiotic susceptibility. ST 3654 was associated (P < 0.0001) with penA/mtrR/porB pattern IX/G45D/G120K,A121D (n = 13/17) and CMRNG (n = 7) or CMTR (n = 6) isolates. Isolates with chromosomal resistance to tetracycline were significantly associated (P < 0.0001) with several STs and penA/mtrR/porB patterns including: ST 3655 (XXII/A-,G45D/G120N,A121N - n = 8/12), ST 921 (pattern IX/G45D/G120D,A121N - n = 6/9), ST 508 (XXII/G45D/G120D,A121N - n = /6), and ST 3656 (pattern XXII/A-,G45D/G120D,A121N - n = 5/6). 24 isolates had higher cefixime MICs (0.03–0.06 mg/L) and included 17 STs with penA pattern IX (n = 17) and mtrR G45D (n = 16) and porB G120K,A121D (n = 12) mutations. Seven of these isolates were associated (P < 0.0001) with ST 3654 (pattern IX/G45D/G120K,A121D). Conclusions We identified significant associations between particular mutation pattern combinations in penA, mtrR and porB and specific STs. This indicates that certain combined mutation patterns may be predictive of antimicrobial susceptibility and useful for molecular diagnosis.