Efflux Pump Antibiotic Binding Site Mutations Are Associated with Azithromycin Nonsusceptibility in Clinical Neisseria gonorrhoeae Isolates

Abstract

contributed equally to this work. Author order was determined both alphabetically and in order of increasing seniority.L yu and Moseng et al. used cryo-electron microscopy to characterize key residues involved in drug binding by mosaic-like MtrD efflux pump alleles in Neisseria gonorrhoeae (1). Isogenic experiments introducing key MtrD substitutions R714G and K823E increased macrolide MICs, leading the authors to predict that nonmosaic MtrD "gonococcal strains bearing both the mtrR promoter and amino acid changes at MtrD positions 714 or 823 could lead to clinically significant levels of Azi nonsusceptibility resistance." We tested this hypothesis by analyzing a global meta-analysis collection of 4,852 N. gonorrhoeae genomes (2). In support of their prediction, we identified clinical isolates with novel nonmosaic MtrD drug binding site substitutions across multiple genetic backgrounds associated with elevated azithromycin MICs (Table 1).Of the 4,852 isolates, 12 isolates contained nonsynonymous mutations at position R714 to amino acid H, L, or C and 7 isolates contained K823 mutations to E or N in the nonmosaic MtrD background. We did not observe substitutions at positions 174, 669, 821, and 825, in line with the authors' demonstration that isogenic mutants at these codons had identical or lowered macrolide MICs. The azithromycin geometric mean TABLE 1 MtrD substitution strains, associated metadata, and resistance allele genotypes a SRA accession no. Reference AZI MIC (g/ml) MtrD allele Cluster b mtrR promoter RplD G70 allele 23S rRNA penA allele ERR1469714 9

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time

“…They did not observe mutations at positions 74, 669, 821 and 825, as found (and tested) by others [24,43]. Interestingly, the R714H/L/C mutations [43,89] correspond to the R717L/Q mutations discussed before (Section 5.1) present in AcrB-Sa of Salmonella clinical isolates from Bangladesh, Pakistan, India and Nepal (Table 9). The location of these mutations (at R714, K823 (and S821)) are all in the PC2 subdomain and face the PBP (Table 6 and Figure 5), which explains the increase in the MICs for macrolides, but, e.g., not for other drugs such as penicillin, ampicillin, ethidium bromide and crystal violet [43].…”

“…Additionally, Ma et al (2020) analyzed 4852 global N. gonorrhoeae genomes. Of these, 12 contained the mutation R714H/L/C, and seven contained the mutation K823E/N [89]. They did not observe mutations at positions 74, 669, 821 and 825, as found (and tested) by others [24,43].…”

“…[77,78] Legionella pneumophila LpeB S824I (I911L, G1158W, F1124 insert) China Azithromycin (macrolide) [90] Bold underlined indicates the recurring "G288" mutation in AcrB-Ec, AcrB-Sa (Salmonella Typhimurium) [80,81], MexY-Pa and AdeJ-Ab. Bold indicates the "R717" mutation recurring in AcrD-Sa (Salmonella Typhi and Paratyphi A) [85][86][87][88]111] and MtrD-Ng [43,89]. Asterisk (*) indicates direct measured increased MICs.…”

“…K→T) found in MexY from Pseudomonas aeruginosa-experimentally evolved strain, implicated in tobramycin resistance[83]. Additionally, K79A in MexY has increased aminoglycoside (paromomycin) resistance[56]; ** (M→V) found in MexB from Pseudomonas aeruginosa-clinical strains from Denmark[5]; *** (R→L/Q) found in AcrB from Salmonella Typhi and Salmonella Paratyphi A-clinical isolates from Bangladesh, Nepal, India and Pakistan, known to cause azithromycin resistance[85][86][87][88]; **** (R→C/G/H/L) and (K→D/E/N) found in MtrD from Neisseria gonorrhoeae-clinical isolates from India, the USA and the EU, known to cause azithromycin resistance[24,43,89]; ***** (S→I) found in LpeB from Legionella pneumophila-spring water isolates from China[90]. Colors: green background, conserved residues compared to AcrB-Ec.…”

Serum Complement Activation by C4BP-IgM Fusion Protein Can Restore Susceptibility to Antibiotics in Neisseria gonorrhoeae

“…The multiple transferable resistance ( mtr ) operon in N. gonorrhoeae encodes membrane proteins that form an energy-dependent efflux pump system (MtrC-MtrD-MtrE), which removes drugs and hydrophobic agents from inside the cells into the extracellular space ( 18 ). Some gonococcal strains acquire resistance to azithromycin by deletions in the mtrR promoter region (MtrR is a repressor of mtr CDE) or gain-of-function mutations in mosaic mtr efflux pump alleles which result in overproduction/overactivation of efflux pump proteins ( 19 , 20 ). Mtr AMR determinants combined with mutations in genes for the penicillin binding protein 2 (PBP2) and Porin (PorB1b) decrease susceptibility to ceftriaxone ( 21 ).…”