Draft Genome Sequences of Three Penicillin-Resistant Neisseria gonorrhoeae Strains Isolated in Cincinnati, Ohio, in 1994

Here, we report the draft genome sequences of three penicillin-resistant Neisseria gonorrhoeae isolates. We include associated data on MICs and genetic relationships to other N. gonorrhoeae strains collected from across the United States. Resistance mutations known to contribute to reduced penicillin susceptibility are annotated in each genome.

P enicillin resistance in Neisseria gonorrhoeae can be gained through two mechanisms, inheritance of a plasmid-borne penicillinase (bla TEM-1 ) or acquisition of chromosomal mutations. However, the complete suite of causative mutations underlying chromosomal resistance has yet to be determined. Contributors have been identified in a chromosomally mediated resistant N. gonorrhoeae strain (CMRNG), FA6140, isolated from an infected individual in Durham, North Carolina, in 1983 (1) and include (i) mutations in penA encoding a penicillin-binding protein 2 (PBP2) with a decreased penicillin acylation rate (2), (ii) a mutation in the mtrCDE efflux pump (mtr) promoter that increases pump expression (3), and (iii) mutations in the porin P1B allele that decrease porin-mediated influx of penicillin (4). An L421P substitution in PBP1, encoded by ponA, also plays a role in decreased susceptibility (5). To contribute to the understanding of mutations that reduce penicillin susceptibility, we sequenced the genomes of three strains with different levels of resistance.
N. gonorrhoeae strains 111, 114, and 151 were isolated from infected individuals in Cincinnati, Ohio, in 1994 and were provided by Joan Knapp at the Centers for Disease Control and Prevention (CDC). Bacteria were cultivated on GC agar base medium supplemented with 1% Kellogg's solution (6) at 37°C in 5% CO 2 . Susceptibility testing was conducted as previously described (7); all isolates were resistant to penicillin, as determined using the Clinical and Laboratory Standards Institute cutoff of $2 mg/ml (8), with MICs from 4 to 32 mg/ml (Table 1).
Cells were grown overnight on agar plates, and genomic DNA was purified using the Thermo Fisher PureLink genomic DNA minikit following lysis in Tris-EDTA buffer with 0.5 mg/ml lysozyme and 3 mg/ml proteinase K. Illumina Nextera XT-prepared libraries were pooled and sequenced using a V3 600-cycle cartridge (2 Â 300 bp) on the Illumina MiSeq platform at the Rochester Institute of Technology Genomics Core. For all analyses, default parameters were used except where otherwise noted. Pairedend sequencing resulted in a total of 7.97 million reads across the three samples, and after poor quality sequences were trimmed using Trimmomatic v.0.39 (9), a total of 7.46 million reads remained. SPAdes v.3.13.0 (10) was used for assembly, statistics were reported using QUAST (11), and genes were annotated with Prokka v.1.14.5 (12) ( Table 1). We identified resistance mutations by alignment to FA6140 (GenBank accession number CP012027.1) and FA19 (CP012026.1) sequences using BLASTn. To identify the phylogenetic placement of new genomes, we reconstructed the phylogeny of 2,652 gonococcal strains isolated in the United States between 1983 and 2016 (13-16) (Fig. 1). An alignment was created by mapping to NCCP11945 (NC_011035.1) (see    (19), and iTOL v.5 (20) were used to construct and visualize the tree. Isolates 111 and 114 had penicillin MICs of 4 and 6 mg/ml, respectively, and did not harbor the b-lactamase gene, suggesting that they are similar to the CMRNG strain FA6140. Indeed, out of the 2,652 strains in our cohort, these isolates were among the nearest phylogenetic neighbors to FA6140 (Fig. 1), despite isolation 11 years later from a distinct geographic location. The number of polymorphic sites from the FA6140 reference genome was 484 and 465 for 111 and 114, respectively. These isolates had the same haplotype of known resistance determinants as FA6140 (Table 1).
Strain 151 had a penicillin MIC of 32 mg/ml, and analysis confirmed the presence of bla TEM-1 , indicating that it is a penicillinase-producing strain. The assembly contained a 5,727-bp contig that differed by only two insertions from the top BLAST hit (GenBank accession number MH140435.1), the African-type pJD5 gonococcal plasmid (21). In addition, 151 had an mtrR A39T substitution, which increases the expression of mtrCDE (22) ( Table 1), and substitutions in penA (Table 1), but these are likely minor contributors to resistance in this strain.
Future comparative analyses of the CMRNG genomes reported in this announcement may help to further illuminate the genetic basis of penicillin resistance in gonococci.
Data availability. The accession numbers for genome assemblies and raw reads are listed in Table 1 and available for download through GenBank and the SRA, respectively. All code is accessible at https://github.com/wadsworthlab.