Jo Anne R. Dillon

Epidemic methicillin-susceptible Staphylococcus aureus lineages are the main cause of infections at an Iranian university hospital.

ABSTRACT The majority of Staphylococcus aureus infections from Isfahan, Iran, were caused by epidemic methicillin-susceptible S. aureus (MSSA) lineages, sequence type 8 (ST8), ST22, ST30, and ST6. The predominant methicillin-resistant S. aureus strain was ST239. We observed a high prevalence of Panton-Valentine leukocidin-positive MSSA strains (19.7%), which is a matter of considerable concern, since these strains have the ability to cause severe infections.

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.

Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance : a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains

ABSTRACT A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca ). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n = 109) were identified as unrelated singletons. NG-STAR had a high Simpsons diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n = 100; 13.0%), ST-42 and ST-91 (n = 45; 5.9%), ST-64 (n = 44; 5.72%), and ST-139 (n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains.

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.

Influence of Conserved and Hypervariable Genetic Markers on Genotyping Circulating Strains of Neisseria gonorrhoeae

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.

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.

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.