scholarly journals Azithromycin resistance through interspecific acquisition of an epistasis dependent efflux pump component and transcriptional regulator in Neisseria gonorrhoeae

2018 ◽  
Author(s):  
Crista B. Wadsworth ◽  
Brian J. Arnold ◽  
Mohamad R. Abdul Sater ◽  
Yonatan H. Grad
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Structural Changes ◽  
Population Genomics ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Full Length ◽  
Mechanism Of Resistance ◽  
Promoter Mutations ◽  
Azithromycin Resistance

ABSTRACTMosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with reduced susceptibility to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hotspot of interspecific recombination introducing alleles from N. meningitidis and N. lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrated that resistance is mediated through epistasis between these two loci and that the full length of the mosaic mtrD allele is required. Gene expression profiling revealed the mechanism of resistance in mosaics couples the novel mtrDalleles with promoter mutations enhancing expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple Neisseria has contributed to azithromycin resistance in the gonococcal population.AUTHOR SUMMARYNeisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for over 100 million cases of gonorrhea worldwide each year. The incidence of reduced susceptibility to the macrolide class antibiotic azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance to this drug remains unexplained. Recently, resistance has been shown to be highly associated with mosaic alleles of the multiple transferable resistance (mtr) efflux pump, which have been gained via horizontal gene exchange with other Neisseria. However, if and how these alleles caused resistance was unknown. Here, we demonstrate that resistance has been gained through epistasis between mtrD and the mtr promoter region using evidence from both population genomics and experimental genetic manipulation. Epistasis also acts within the mtrD locus alone, requiring the full length of the gene for phenotypic resistance. Transcriptomic profiling indicates that the mechanism of resistance in mosaics is likely derived from both structural changes to mtrD, coupled with promoter mutations that result in regulatory changes to mtrCDE.

scholarly journals Azithromycin Resistance through Interspecific Acquisition of an Epistasis-Dependent Efflux Pump Component and Transcriptional Regulator inNeisseria gonorrhoeae

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Crista B. Wadsworth ◽  
Brian J. Arnold ◽  
Mohamad R. Abdul Sater ◽  
Yonatan H. Grad
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antimicrobial Agents ◽  
Population Genomics ◽  
Efflux Pump ◽  
Hot Spot ◽  
Bacterial Pathogen ◽  
Epidemiological Studies ◽  
Content Type ◽  
And Control ◽  
Azithromycin Resistance

ABSTRACTMosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin inNeisseria gonorrhoeaein epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variantmtralleles and azithromycin resistance. We find that the locus encompassing themtrRtranscriptional repressor and themtrCDEpump is a hot spot of interspecific recombination introducing alleles fromNeisseria meningitidisandNeisseria lactamicaintoN. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium atmtrDand themtrpromoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaicmtrDallele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novelmtrDalleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions atmtrgained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents.IMPORTANCENeisseria gonorrhoeaeis the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance throughmtralleles from otherNeisseriaspecies, demonstrating that commensalNeisseriabacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2021 ◽  
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.

scholarly journals Increased antibiotic susceptibility in Neisseria gonorrhoeae through adaptation to the cervical environment

2020 ◽  
Author(s):  
Kevin C Ma ◽  
Tatum D Mortimer ◽  
Allison L Hicks ◽  
Nicole E Wheeler ◽  
Leonor Sánchez-Busó ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antibiotic Susceptibility ◽  
Microbial Population ◽  
Population Genomics ◽  
Efflux Pump ◽  
Loss Of Function ◽  
Environmental Pressures ◽  
Genome Wide ◽  
A Subunit ◽  
The Impact

AbstractNeisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association on the largest collection of N. gonorrhoeae isolates to date (n=4852) to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical isolates relative to urethral isolates (odds ratio (OR) = 3.74, 95% CI [1.98-6.70]). In support of a model in which pump expression incurs a fitness cost in this niche, cervical isolates were also enriched relative to urethral isolates in LOF mutations in the mtrCDE activator mtrA (OR = 8.60, 95% CI [4.96-14.57]) and in farA, a subunit of the FarAB efflux pump (OR = 6.25, 95% CI [3.90-9.83]). In total, approximately 2 in 5 cervical isolates (42.6%) contained a LOF mutation in either the efflux pump components mtrC or farA or the activator mtrA. Our findings extend beyond N. gonorrhoeae to other Neisseria: mtrC LOF mutations are rare (<1%) in the primarily nasopharyngeal-colonizing N. meningitidis in a collection of 14,798 genomes but enriched in a heterosexual urethritis-associated lineage (8.6%, p = 9.90×10−5), indicating that efflux pump downregulation contributes broadly to the adaptation of pathogenic Neisseria to the female urogenital tract. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.

scholarly journals Mutation in 23S rRNA Associated with Macrolide Resistance in Neisseria gonorrhoeae

2002 ◽  
Vol 46 (9) ◽  
pp. 3020-3025 ◽  
Author(s):  
Lai-King Ng ◽  
Irene Martin ◽  
Gary Liu ◽  
Louis Bryden
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Promoter Region ◽  
Efflux Pump ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
E Coli ◽  
Single Base Pair ◽  
Resistant Strains ◽  
Domain V

ABSTRACT Fifty-six azithromycin-resistant (MICs, 2.0 to 4.0 μg/ml) Neisseria gonorrhoeae strains with cross-resistance to erythromycin (MICs, 2.0 to 64.0 μg/ml), isolated in Canada between 1997 and 1999, were characterized, and their mechanisms of azithromycin resistance were determined. Most (58.9%) of them belonged to auxotype-serotype class NR/IB-03, with a 2.6-mDa plasmid. Based on resistance to crystal violet (MICs ≥ 1 μg/ml), 96.4% of these macrolide-resistant strains appeared to have increased efflux. Nine of the eleven strains selected for further characterization were found to have a promoter region mtrR mutation, a single-base-pair (A) deletion in the 13-bp inverted repeat, which is believed to cause overexpression of the mtrCDE-encoded efflux pump. The two remaining macrolide-resistant strains (erythromycin MIC, 64.0 μg/ml; azithromycin MIC, 4.0 μg/ml), which did not have the mutation in the mtrR promoter region, were found to have a C2611T mutation (Escherichia coli numbering) in the peptidyltransferase loop in domain V of the 23S rRNA alleles. Although mutations in domain V of 23S rRNA alleles had been reported in other bacteria, including E. coli, Streptococcus pneumoniae, and Helicobacter pylori, this is the first observation of these mutations associated with macrolide resistance in N. gonorrhoeae.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2020 ◽  
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

AbstractObjectivesThe prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. The aim of this study was to characterize the genetic pathways leading to high-level azithromycin resistance.MethodsA 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.ResultsWithin 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-encoded 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.ConclusionsThis 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.

scholarly journals 1440. The Genetic Basis for a Neisseria gonorrhoeae Clinical Isolate That Contains mtrR-79 Mutation But Is Highly Susceptible to Antibiotics Effluxed by the Mtr Pump System

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S724-S724
Author(s):  
Jianzhong Huang ◽  
Karen Ingraham
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Clinical Isolate ◽  
Promoter Region ◽  
Genetic Basis ◽  
Natural Transformation ◽  
Efflux Pump ◽  
Multiple Drug ◽  
Loss Of Function ◽  
Pump System ◽  
Susceptible Isolate

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)

scholarly journals Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Anmoldeep Randhawa ◽  
Nandita Pasari ◽  
Tulika Sinha ◽  
Mayank Gupta ◽  
Anju M. Nair ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Engineering ◽  
Efflux Pump ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Drug Tolerance ◽  
Growth Media ◽  
Penicillium Funiculosum ◽  
Cellobiohydrolase I ◽  
Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

scholarly journals Overexpression of the MtrC-MtrD-MtrE Efflux Pump Due to an mtrR Mutation Is Required for Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae

2002 ◽  
Vol 184 (20) ◽  
pp. 5619-5624 ◽  
Author(s):  
Wendy L. Veal ◽  
Robert A. Nicholas ◽  
William M. Shafer
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Base Pair ◽  
Efflux Pump ◽  
Penicillin Resistance ◽  
Single Base ◽  
Base Pair Deletion ◽  
Resistance To Penicillin ◽  
Single Base Pair ◽  
High Level ◽  
Level Resistance

ABSTRACT The importance of the mtrCDE-encoded efflux pump in conferring chromosomally mediated penicillin resistance on certain strains of Neisseria gonorrhoeae was determined by using genetic derivatives of penicillin-sensitive strain FA19 bearing defined mutations (mtrR, penA, and penB) donated by a clinical isolate (FA6140) expressing high-level resistance to penicillin and antimicrobial hydrophobic agents (HAs). When introduced into strain FA19 by transformation, a single base pair deletion in the mtrR promoter sequence from strain FA6140 was sufficient to provide high-level resistance to HAs (e.g., erythromycin and Triton X-100) but only a twofold increase in resistance to penicillin. When subsequent mutations in penA and porIB were introduced from strain FA6140 into strain WV30 (FA19 mtrR) by transformation, resistance to penicillin increased incrementally up to a MIC of 1.0 μg/ml. Insertional inactivation of the gene (mtrD) encoding the membrane transporter component of the Mtr efflux pump in these transformant strains and in strain FA6140 decreased the MIC of penicillin by 16-fold. Genetic analyses revealed that mtrR mutations, such as the single base pair deletion in its promoter, are needed for phenotypic expression of penicillin and tetracycline resistance afforded by the penB mutation. As penB represents amino acid substitutions within the third loop of the outer membrane PorIB protein that modulate entry of penicillin and tetracycline, the results presented herein suggest that PorIB and the MtrC-MtrD-MtrE efflux pump act synergistically to confer resistance to these antibiotics.

Sign in / Sign up

Export Citation Format

Share Document