Exploration of theNeisseriaresistome reveals resistance mechanisms in commensals that may be acquired byN. gonorrhoeaethrough horizontal gene transfer

Abstract

Non-pathogenic Neisseria have repeatedly been demonstrated to transfer antibiotic resistance genes to their pathogenic relative, Neisseria gonorrhoeae. However, the resistance genotypes and subsequent phenotypes of non-pathogens within the genus have been studied and described less frequently. Here, we use Etests to characterize the minimum inhibitory concentrations (MICs) of a panel of Neisseria (n=26) - including several commensal species - acquired from the CDC & FDA's Antibiotic Resistance (AR) Isolate Bank to a suite of diverse antibiotics. We furthermore use whole genome sequencing and the Comprehensive Antibiotic Resistance Database (CARD) Resistance Gene Identifier (RGI) platform to predict possible causal resistance-encoding mutations. Within this panel, resistant isolates to all tested antimicrobials including penicillin (n=5/26), ceftriaxone (n=2/26), cefixime (n=3/26), tetracycline (n=10/26), azithromycin (n=11/26), and ciprofloxacin (n=4/26) were found. In total we identify 63 distinct mutations predicted by RGI to be involved in resistance. The presence of several of these mutations had clear associations with increases in MIC such as: DNA gyrase subunit A (gyrA) (S91F) and ciprofloxacin, tetracycline resistance protein (tetM) and 30S ribosomal protein S10 (rpsJ) (V57M) and tetracycline, and TEM-type beta-lactamases and penicillin. However, mutations with strong associations to macrolide and cephalosporin resistance were not conclusive. This work serves as an initial exploration into the resistance-encoding mutations harbored by non-pathogenic Neisseria, which will ultimately aid in prospective surveillance for novel resistance mechanisms that may be rapidly acquired by N. gonorrhoeae.

Constrained evolutionary paths to macrolide resistance in aNeisseriacommensal converge on ribosomal genes through sequence duplication

“…Overall, our results expand on prior studies that provide initial insights into illuminating the commensal resistome (17,21,22) , which is a known source of antibiotic resistance for Neisseria pathogens (6,(8)(9)(10). We find evidence of constraint on high level macrolide resistance genotypic evolution in AR-0945, with convergence on only two sites in the genome, however different genetic starting places will likely impact evolutionary outcome due to epistatic and additive effects between loci.…”

“…Evolutionary trajectories to macrolide resistance in N. elongata N. elongata AR Bank #0945 was selected to explore the evolutionary paths to azithromycin resistance in a Neisseria commensal. N. elongata AR Bank #0945 has been tested for its minimum inhibitory concentration (MIC) to azithromycin (0.38 μ g/mL) and sequenced (SAMN15454046) by our group previously (21). In this study, a draft genome was assembled as an ancestral reference for all derived lineages.…”

“…Multiple studies have demonstrated that commensal Neisseria serve as reservoirs of resistance for Ngo (6,(8)(9)(10), however a comprehensive evaluation of the resistance alleles commensals can harbor, though underway (17,21,22), and their likelihood of transfer to pathogenic relatives is still in its infancy. Here, we use experimental evolution to screen for the mutations that impart azithromycin resistance in Nel and assess their potential for transfer to Ngo.…”

“…Additionally, the majority of resistance to third-generation cephalosporins in gonococci is derived through mosaic penicillin-binding protein 2 (penA) alleles gained from close commensal relatives (6,10). Though studies that characterize the resistance genotypes and phenotypes in panels of commensal Neisseria (such as (17,21,22)) will aid in prospective surveillance for novel resistance that may be rapidly acquired by Ngo, the utility of these approaches is ultimately limited by extensive under sampling of natural commensal populations.…”

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

“…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 μg/ml ( 8 ), with MICs from 4 to 32 μg/ml ( Table 1 ).…”