A Novel Method to Assess Antimicrobial Susceptibility in Commensal Oropharyngeal Neisseria—A Pilot Study

Abstract

Commensal Neisseria provide a reservoir of resistance genes that can be transferred to the pathogens Neisseria gonorrhoeae and N. meningitidis in the human oropharynx. Surveillance programs are thus needed to monitor resistance in oropharyngeal commensal Neisseria, but currently the isolation and antimicrobial susceptibility testing of these commensals is laborious, complex and expensive. In addition, the posterior oropharyngeal/tonsillar swab, which is commonly used to sample oropharyngeal Neisseria, is poorly tolerated by many individuals. We evaluated an alternative non-invasive method to isolate oropharyngeal commensal Neisseria and to detect decreased susceptibility to azithromycin using selective media (LBVT.SNR) with and without azithromycin (2 µg/mL). In this pilot study, we compared paired posterior oropharyngeal/tonsillar swabs and oral rinse-and-gargle samples from 10 participants and demonstrated that a similar Neisseria species diversity and number of colonies were isolated from both sample types. Moreover, the proportion of Neisseria colonies that had a decreased susceptibility to azithromycin was similar in the rinse samples compared to the swabs. This pilot study has produced encouraging data that a simple protocol of oral rinse-and-gargle and culture on plates selective for commensal Neisseria with and without a target antimicrobial can be used as a surveillance tool to monitor antimicrobial susceptibility in commensal oropharyngeal Neisseria. Larger studies are required to validate these findings.

Canary in the Coal Mine: How Resistance Surveillance in Commensals Could Help Curb the Spread of AMR in Pathogenic Neisseria

“…2 ). To achieve this, we recommend prioritizing oropharyngeal samples, which are more likely to harbor commensals than rectal samples, through oral rinse-and-gargle to collect diverse Neisseria species from distinct ecological sites in the mouth and nasopharynx, as described by Laumen et al ( 138 ). The oral rinse-and-gargle method has been demonstrated to capture increased Neisseria species diversity as opposed to a posterior oropharyngeal/tonsillar swab, is less invasive than a swab, and is more readily implementable as a standardized tool across clinics ( 138 ).…”

“…Retained colonies are then subjected to AMR profiling and WGS. We propose a minor modification to the eGISP program (bold and red), in which all pharyngeal samples collected via oral rinse-and-gargle ( 138 ) are conserved if they display features inconsistent with N. gonorrhoeae , yet consistent with other members of the Neisseria genus (Gram−, diplococci or rods, and oxidase+) (bold and red). We also suggest that pharyngeal samples are inoculated onto LBVT.SNR media, which is selective for commensal Neisseria.…”

“…However, commensal Neisseria display variable sensitivity to colistin which limits the utility of MTM media for sampling commensals ( 139 , 140 ). Therefore, we suggest the use of an additional plate of complementary medium, specifically designed to isolate commensal Neisseria (LB containing vancomycin, trimethoprim, sucrose, neutral red, LBVT.SNR) ( 19 , 138 ). While labor-intensive, this new strategy will undoubtedly create a wider and more diverse collection of commensal Neisseria than is currently available.…”

“…Once isolates are obtained, AMR profiling and WGS bioinformatics workflows used with N. gonorrhoeae can be expanded to newly isolated commensal Neisseria ( 142 , 143 ). Alternatively, low-cost semiquantitative AMR profiling, by adding antibiotics of interest at an established concentration to plates, could be used to further select for AMR Neisseria ( 138 ). WGS and AMR profiling data can be used as the input for established bioinformatics pipelines.…”

“…Therefore, the surveillance of commensal Neisseria species would require minor changes in the methodology of the clinical microbiology lab, and would not modify patients’ experiences nor sample collection at large ( 138 ). We acknowledge that our proposed modification to eGISP (adding the collection, characterization, and storage of commensal isolates) will be both a costly endeavor and require significantly increased effort for the personnel involved in surveillance programs; however, we also believe that our current blindness of known AMR reservoirs for Neisseria pathogens is a key gap in addressing AMR within the genus.…”