Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

RluA is the major mRNA pseudouridine synthase in Escherichia coli.

Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life. Conserved pseudouridine synthases modify the mRNAs of diverse eukaryotes, but the modification has yet to be identified in bacterial mRNAs. Here, we report the discovery of pseudouridines in mRNA from E. coli. By testing the mRNA modification capacity of all 11 known pseudouridine synthases, we identify RluA as the predominant mRNA-modifying enzyme. RluA, a known tRNA and 23S rRNA pseudouridine synthase, modifies at least 31 of the 44 high-confidence sites we identified in E. coli mRNAs. Using RNA structure probing data to inform secondary structures, we show that the target sites of RluA occur in a common sequence and structural motif comprised of a ΨURAA sequence located in the loop of a short hairpin. This recognition element is shared with previously identified target sites of RluA in tRNAs and rRNA. Overall, our work identifies pseudouridine in key mRNAs and suggests the capacity of Ψ to regulate the transcripts that contain it.

The Bacillus subtilis ywbD gene encodes RlmQ, the 23S rRNA methyltransferase forming m7G2574 in the A-site of the peptidyl transferase center.

Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis, the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m7G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m7G methyltransferase candidates allowed us to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis.

RlmQ: a newly discovered rRNA modification enzyme bridging RNA modification and virulence traits in Staphylococcus aureus.

rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analyses of the rRNA modifications in taxonomically distant bacteria could help define their general, as well as species-specific, roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in Escherichia coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralog of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.

Identification of leader-trailer helices of precursor ribosomal RNA in all phyla of bacteria and archaea.

Ribosomal RNAs are transcribed as part of larger precursor molecules. In Escherichia coli, complementary RNA segments flank each rRNA and form long leader-trailer (LT) helices, which are crucial for subunit biogenesis in the cell. A previous study of 15 representative species suggested that most but not all prokaryotes contain LT helices. Here, we use a combination of in silico folding and covariation methods to identify and characterize LT helices in 4464 bacterial and 260 archaeal organisms. Our results suggest that LT helices are present in all phyla, including Deinococcota, which had previously been suspected to lack LT helices. In very few organisms, our pipeline failed to detect LT helices for both 16S and 23S rRNA. However, a closer case-by-case look revealed that LT helices are indeed present but escaped initial detection. Over 3600 secondary structure models, many well supported by nucleotide covariation, were generated. These structures show a high degree of diversity. Yet, all exhibit extensive base-pairing between the leader and trailer strands, in line with a common and essential function.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it.

The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.

Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. Bacillus subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after Escherichia coli and Thermus thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli.

Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Roles of the leader-trailer helix and antitermination complex in biogenesis of the 30S ribosomal subunit.

Ribosome biogenesis occurs co-transcriptionally and entails rRNA folding, ribosomal protein binding, rRNA processing, and rRNA modification. In most bacteria, the 16S, 23S and 5S rRNAs are co-transcribed, often with one or more tRNAs. Transcription involves a modified RNA polymerase, called the antitermination complex, which forms in response to cis-acting elements (boxB, boxA and boxC) in the nascent pre-rRNA. Sequences flanking the rRNAs are complementary and form long helices known as leader-trailer helices. Here, we employed an orthogonal translation system to interrogate the functional roles of these RNA elements in 30S subunit biogenesis in Escherichia coli. Mutations that disrupt the leader-trailer helix caused complete loss of translation activity, indicating that this helix is absolutely essential for active subunit formation in the cell. Mutations of boxA also reduced translation activity, but by only 2- to 3-fold, suggesting a smaller role for the antitermination complex. Similarly modest drops in activity were seen upon deletion of either or both of two leader helices, termed here hA and hB. Interestingly, subunits formed in the absence of these leader features exhibited defects in translational fidelity. These data suggest that the antitermination complex and precursor RNA elements help to ensure quality control during ribosome biogenesis.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Novel Variant and Known Mutation in 23S rRNA Gene of Mycoplasma pneumoniae, Northern Vietnam, 2023.

During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.

Mycoplasmoides genitalium nucleic acid semi-quantitation and molecular macrolide resistance detection via automated assays: gender and specimen source considerations.

A laboratory-developed test (LDT) using analyte-specific reagents has been optimized on a commercial platform to detect macrolide resistance-associated mutations (MRM) in 23S rRNA from Mycoplasmoides genitalium from primary clinical specimens. In this study, MRM-LDT was applied to a multi-specimen source study set. One thousand four hundred ninety-five primary specimens testing positive for M. genitalium by commercial transcription-mediated amplification (TMA) were initially titered by the TMA assay using serial 10-fold dilutions to semi-quantitate target nucleic acid burden. Primary specimens were then processed for MRM detection using the MRM-LDT. Findings were stratified by gender and specimen source. The mean log10 target nucleic acid titer of a TMA-positive specimen was 3.51 (median 3; range 0-10). Male specimens (n = 1145) demonstrated a mean log10 M. genitalium TMA titer of 3.67; that value observed in 350 female specimens was 2.98 (P < 0.0001). The MRM-LDT detection rate (88.7%) from specimens with log10 M. genitalium TMA titers ≥ 4 was increased over specimens with log10 titers ≤ 1 (4.5%; P < 0.0002). In females, MRM-LDT was positive from 51.3% of vaginal swab and 34.7% of urine specimens (P = 0.01). In males, MRM-LDT was positive from 65.0% of rectal swab and 55.7% of urine specimens (P = 0.002). Differences were also observed in log10 M. genitalium TMA titers as a function of specimen source. M. genitalium macrolide resistance rates among multiple specimen sources, as determined by MRM-LDT, are high in the United States and can be consistent with target nucleic acid burden within the primary specimen. Caveats experienced within subgroupings support MRM reflex testing on primary M. genitalium-positive specimens. IMPORTANCE: First-line macrolide treatment failure is of increasing concern with Mycoplasmoides genitalium in multiple settings. Recent sexually-transmitted infection treatment guidelines from the United States Centers for Disease Control and Prevention have predicated therapeutic approaches on the availability of a macrolide resistance/susceptibility result from a primary clinical specimen. In this report, we investigate potential correlation between macrolide resistance mutation detection rates (identified by a molecular amplified laboratory-developed test) and transcription-mediated amplification-based rRNA target semi-quantitation. Data reveal that rRNA semi-quantitation and laboratory-developed test detection rate differences exist as a function of gender and specimen source. These data can guide providers in proper specimen selection not only for the laboratory diagnosis of M. genitalium but also macrolide resistance mutation determination from primary clinical specimens.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.

Genetic basis of macrolide resistance in porcine Pasteurella multocida isolates from the German national resistance monitoring program GERM-Vet 2008-2021.

OBJECTIVES: To analyse porcine Pasteurella multocida isolates obtained from the national resistance monitoring program GERM-Vet 2008-2021 in Germany for phenotypic and genotypic macrolide resistance. METHODS: The antimicrobial susceptibility of the isolates was determined by broth microdilution according to CLSI standards. Closed genomes were obtained by WGS via Illumina MiSeq and MinION platforms followed by a hybrid assembly. RESULTS: Of 1114 porcine P. multocida isolates, only four isolates (0.36%), one each from 2008, 2010, 2019 and 2021, exhibited resistance to at least one macrolide tested. The isolate from 2010 was only resistant to erythromycin and WGS analysis neither revealed a macrolide resistance gene nor a macrolide resistance-mediating mutation. The isolates from 2008 and 2019 were resistant to erythromycin, tilmicosin, tildipirosin, tulathromycin and gamithromycin and showed either only the A2058G mutation in all six 23S rRNA operons or the chromosomally located macrolide resistance genes msr(E) and mph(E), respectively. The isolate from 2021 was resistant to erythromycin, tulathromycin, gamithromycin and tylosin and carried a novel integrative and conjugative element of 64 966 bp, designated Tn7730, in its chromosomal DNA. It harboured the macrolide resistance genes mef(C), mph(G) and estT, the lincosamide resistance gene lnu(H), and the tetracycline resistance gene tet(Y), the last two were detected for the first time in P. multocida. CONCLUSION: Macrolide resistance in German porcine P. multocida can be due to resistance-mediating mutations or resistance genes. The presence of the novel Tn7730 carrying three different macrolide resistance genes is alarming and should be monitored.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.