Faculty Opinions recommendation of Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus.

Abstract Background Neisseria gonorrhoeae (NG) possesses multiple drug efflux systems that play an important role in evading antibiotics in the treatment for gonorrhea and in helping this pathogen to evade innate antimicrobial defenses during infection. The mtrR-79 and mtr120 mutations in the promoter region between mtrR and mtrCDE are common mutations contributing to overexpression of the MtrCDE efflux pump resulting in increased efflux to multiple antibiotics including macrolides, β-lactams and tetracycline. However, we found a NG clinical isolate that contains the mtrR-79 mutation but is highly susceptible to antibiotics effluxed by the MtrCDE pump system. Methods PCR amplification, DNA sequencing and natural transformation were used to investigate the genetic basis responsible for the increased susceptibility by this isolate. Results We amplified by PCR the individual genes of mtrCDE, respectively, from this susceptible isolate as well as a NG isolate that contains the mtrR-79 mutation with increased efflux; there was no difference in the size of PCR products between the susceptible isolate and the isolate with increased efflux, indicating there was no large deletion/insertion in these genes. DNA sequence analysis of mtrCDE revealed the susceptible isolate also contained a loss-of-function mutation ΔGC from a 6 GC repeat GCGCGCGCGCGC in mtrC resulting in MtrC A117 frameshift predicted to produce a truncated MtrC protein that results in a low efflux phenotype. Natural transformation of the susceptible isolate with a wild type mtrC and selection with ciprofloxacin generated transformants that corrected the ΔGC mutation and restored the increased efflux phenotype. Conclusion Our results indicate that genotyping of mtrR and the promoter region between mtrR and mtrCDE is insufficient to predict increased efflux phenotype and provide direct evidence that NG isolates with elevated efflux is able to genetically revert to low efflux via loss-of-function mutations in the coding region of the efflux pump genes. Disclosures Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder)

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Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate ofNeisseria gonorrhoeaePossessing a Mosaic-LikemtrEfflux Pump Locus

mBio ◽

10.1128/mbio.02281-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 26

Author(s):

Corinne E. Rouquette-Loughlin ◽

Jennifer L. Reimche ◽

Jacqueline T. Balthazar ◽

Vijaya Dhulipala ◽

Kim M. Gernert ◽

...

Keyword(s):

Amino Acid ◽

Antimicrobial Susceptibility ◽

Dna Sequences ◽

Efflux Pump ◽

Spontaneous Mutation ◽

Content Type ◽

Transporter Protein ◽

Single Nucleotide Change ◽

Clinical Resistance ◽

Mechanistic Basis

ABSTRACTRecent reports suggest that mosaic-like sequences within themtr(multipletransferableresistance) efflux pump locus ofNeisseria gonorrhoeae, likely originating from commensalNeisseriasp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within themtrRgene encoding the transcriptional repressor (MtrR) of themtrCDEefflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018,https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strongcis-acting transcriptional impact of a single nucleotide change within the −35 hexamer of themtrCDEpromoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-likemtrlocus.IMPORTANCEHistorically, after introduction of an antibiotic for treatment of gonorrhea, strains ofN. gonorrhoeaeemerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of theNeisseriagenus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensalNeisseriathat recombine within the gonococcalmtrefflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

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Mechanistic basis for decreased antimicrobial susceptibility in a clinical isolate ofNeisseria gonorrhoeaepossessing a mosaic-likemtrefflux pump locus

10.1101/448712 ◽

2018 ◽

Author(s):

Corinne E. Rouquette-Loughlin ◽

Jennifer L. Reimche ◽

Jacqueline T. Balthazar ◽

Vijaya Dhulipala ◽

Kim M. Gernert ◽

...

Keyword(s):

Amino Acid ◽

Antimicrobial Susceptibility ◽

Dna Sequences ◽

Efflux Pump ◽

Spontaneous Mutation ◽

Gene Encoding ◽

Transporter Protein ◽

Single Nucleotide Change ◽

Clinical Resistance ◽

Mechanistic Basis

AbstractRecent reports suggest that mosaic-like sequences within themtr(multipletransferableresistance) efflux pump locus ofNeisseria gonorrhoeaelikely originating from commensalNeisseria sp.by transformation can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within themtrRgene encoding the transcriptional repressor (MtrR) of themtrCDEefflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (Wadsworthet al.2018. MBio. doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates including Azi. We report that a strongcis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of themtrCDEpromoter as well gain-of-function amino acid changes at theC-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-likemtrlocus.IMPORTANCEHistorically, after introduction of an antibiotic for treatment of gonorrhea, strains ofN. gonorrhoeaeemerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of theNeisseriagenus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented herein provide a framework for understanding how mosaic-like DNA sequences from commensalNeisseriathat recombine within the gonococcalmtrefflux pump locus function to decrease bacterial susceptibility to antimicrobials including antibiotics used in therapy of gonorrhea.

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Expanding U.S. Laboratory Capacity for Neisseria gonorrhoeae Antimicrobial Susceptibility Testing and Whole-Genome Sequencing through the CDC's Antibiotic Resistance Laboratory Network

Journal of Clinical Microbiology ◽

10.1128/jcm.01461-19 ◽

2020 ◽

Vol 58 (4) ◽

Cited By ~ 2

Author(s):

Ellen N. Kersh ◽

Cau D. Pham ◽

John R. Papp ◽

Robert Myers ◽

Richard Steece ◽

...

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Neisseria Gonorrhoeae ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Whole Genome ◽

Content Type ◽

Laboratory Capacity

ABSTRACT U.S. gonorrhea rates are rising, and antibiotic-resistant Neisseria gonorrhoeae (AR-Ng) is an urgent public health threat. Since implementation of nucleic acid amplification tests for N. gonorrhoeae identification, the capacity for culturing N. gonorrhoeae in the United States has declined, along with the ability to perform culture-based antimicrobial susceptibility testing (AST). Yet AST is critical for detecting and monitoring AR-Ng. In 2016, the CDC established the Antibiotic Resistance Laboratory Network (AR Lab Network) to shore up the national capacity for detecting several resistance threats including N. gonorrhoeae. AR-Ng testing, a subactivity of the CDC’s AR Lab Network, is performed in a tiered network of approximately 35 local laboratories, four regional laboratories (state public health laboratories in Maryland, Tennessee, Texas, and Washington), and the CDC’s national reference laboratory. Local laboratories receive specimens from approximately 60 clinics associated with the Gonococcal Isolate Surveillance Project (GISP), enhanced GISP (eGISP), and the program Strengthening the U.S. Response to Resistant Gonorrhea (SURRG). They isolate and ship up to 20,000 isolates to regional laboratories for culture-based agar dilution AST with seven antibiotics and for whole-genome sequencing of up to 5,000 isolates. The CDC further examines concerning isolates and monitors genetic AR markers. During 2017 and 2018, the network tested 8,214 and 8,628 N. gonorrhoeae isolates, respectively, and the CDC received 531 and 646 concerning isolates and 605 and 3,159 sequences, respectively. In summary, the AR Lab Network supported the laboratory capacity for N. gonorrhoeae AST and associated genetic marker detection, expanding preexisting notification and analysis systems for resistance detection. Continued, robust AST and genomic capacity can help inform national public health monitoring and intervention.

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Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab084 ◽

2021 ◽

Cited By ~ 1

Author(s):

J G E Laumen ◽

S S Manoharan-Basil ◽

E Verhoeven ◽

S Abdellati ◽

I De Baetselier ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Ribosomal Proteins ◽

Efflux Pump ◽

23S Rrna ◽

Rrna Gene ◽

23S Rrna Gene ◽

Evolution Of Resistance ◽

High Level ◽

Azithromycin Resistance ◽

Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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