Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES - August, 2019

Neisseria gonorrhoeae antimicrobial resistance (AMR) monthly UPDATES

August, 2019

STI & HIV World Congress - Conference Abstracts

Joint Meeting of the 23rd ISSTDR and 20th IUSTI

July 14–17, 2019 Vancouver, Canada

Editors: David Lewis, James Blanchard, Caroline Cameron, Charlotte Gaydos

 

Webinar

A Call to Action: Responding to Antibiotic Resistant Gonorrhea

Greta Anuschuetz, MPH; Vicki Wilkey, RN, MSN; Jaime Black, MPH

Health Care Education & Training (HCET) [Webinar]. Recorded 2019 June 25.

 

Epidemiology and Surveillance

The impact of antimicrobials on gonococcal evolution

Sánchez-Busó L, Golparian D, Corander J, Grad YH, Ohnishi M, Flemming R, Parkhill J, Bentley SD, Unemo M, Harris SR. (Full Text)

Nat Microbiol. 2019 Jul 29. doi: 10.1038/s41564-019-0501-y. [Epub ahead of print]

Abstract

The sexually transmitted pathogen Neisseria gonorrhoeae is regarded as being on the way to becoming an untreatable superbug. Despite its clinical importance, little is known about its emergence and evolution, and how this corresponds with the introduction of antimicrobials. We present a genome-based phylogeographical analysis of 419 gonococcal isolates from across the globe. Results indicate that modern gonococci originated in Europe or Africa, possibly as late as the sixteenth century and subsequently disseminated globally. We provide evidence that the modern gonococcal population has been shaped by antimicrobial treatment of sexually transmitted infections as well as other infections, leading to the emergence of two major lineages with different evolutionary strategies. The well-described multidrug-resistant lineage is associated with high rates of homologous recombination and infection in high-risk sexual networks. A second, multisusceptible lineage is more associated with heterosexual networks, with potential implications for infection control.

Resistance of Neisseria gonorrhoeae isolates to beta-lactam antibiotics (benzylpenicillin and ceftriaxone) in Russia, 2015-2017

Shaskolskiy B1, Dementieva E1, Kandinov I1, Filippova M1, Petrova N2, Plakhova X2, Chestkov A2, Kubanov A2, Deryabin D2, Gryadunov D1. (Full Text)

PLoS One. 2019 Jul 25;14(7):e0220339. doi: 10.1371/journal.pone.0220339. eCollection 2019.

Abstract

The goal of this work was to study the phenotypic susceptibility and resistance determinants of N. gonorrhoeae isolates to beta-lactam antimicrobials (benzylpenicillin and ceftriaxone). A total of 522 clinical isolates collected in Russia in 2015-2017 were analysed for susceptibility using the agar dilution method. DNA loci involved in antimicrobial resistance were identified using DNA microarray analysis and sequencing. Resistance to benzylpenicillin remained high, with 7.7% of isolates resistant (MICpen > 1 mg/L) and 47.5% of isolates showing intermediate susceptibility (MICpen = 0.12-1 mg/L). The most frequent resistance determinant (72.4% isolates) was the Asp345 insertion in penA, both as a single mutation and in combination with other mutations, particularly with the substitution Leu421Pro in ponA (39.0%). Mutations affecting the influx and efflux of drugs were also found, including amino acid substitutions in PorB (26.8% isolates) and delA in the promoter region of mtrR (22.8%). The accumulation of mutations in chromosomal genes (penA, pon, porA, and mtrR) led to a stepwise increase in MICpen to values characteristic of intermediate resistance. The presence of blaTEM plasmids was found in 25 isolates (4.8%), resulting in a strong increase in resistance to penicillin (MICpen > 16 mg/L) compared with the chromosomal mutations; 23 plasmids were of the African type with TEM-1 beta-lactamase, and two plasmids were of the Toronto/Rio type with TEM-135 beta-lactamase. Only three isolates were found with reduced susceptibility to ceftriaxone, with MICcef = 0.12-0.25 mg/L. Sequencing of penA did not reveal mutations associated with resistance to third-generation cephalosporins, and the gene structure was non-mosaic. The majority of isolates (21 of 25) carrying the blaTEM plasmid also contained the conjugative plasmid with tetM (resistance to tetracyclines), consistent with previously reported data that the presence of the conjugative plasmid facilitates the transfer of other plasmids associated with antimicrobial resistance.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae: a Cross-Sectional Study of Isolates Recovered from Routine Urine Cultures in a High-Incidence Setting.

Bailey AL, Potter RF, Wallace MA, Johnson C, Dantas G, Burnham CA. (Full Text)

mSphere. 2019 Jul 24;4(4). pii: e00373-19. doi: 10.1128/mSphere.00373-19.

Abstract

The objectives of this study were to perform genomic and phenotypic characterization of antimicrobial resistance in Neisseria gonorrhoeae isolates recovered from urine samples from patients in St. Louis, MO, USA. Sixty-four clinical isolates were banked over a 2-year period and subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion (penicillin, tetracycline, cefuroxime, and ciprofloxacin) and gradient diffusion (tetracycline, doxycycline, azithromycin, ceftriaxone, cefixime, ciprofloxacin, gemifloxacin, and delafloxacin). The medical records for the patients were evaluated to determine the demographics, location, and prescribed treatment regimen. Isolate draft genomes were assembled from Illumina shotgun sequencing data, and resistance determinants were identified by ResFinder and PointFinder. Of the 64 isolates, 97% were nonsusceptible to penicillin, with resistant isolates all containing the bla TEM-1b gene; 78 and 81% of isolates were nonsusceptible to tetracycline and doxycycline, respectively, with resistant isolates all containing the tet(M) gene. One isolate was classified as non-wild-type to azithromycin, and all isolates were susceptible to ceftriaxone; 89% of patients received this combination of drugs as first-line therapy. Six percent of isolates were resistant to ciprofloxacin, with most resistant isolates containing multiple gyrA and parC mutations. Correlation between disk and gradient diffusion AST devices was high for tetracycline and ciprofloxacin (R2 > 99% for both). The rates of N. gonorrhoeae antibiotic resistance in St. Louis are comparable to current rates reported nationally, except ciprofloxacin resistance was less common in our cohort. Strong associations between specific genetic markers and phenotypic susceptibility testing hold promise for the utility of genotype-based diagnostic assays to guide directed antibiotic therapy.



IMPORTANCE Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is most commonly diagnosed using a DNA-based detection method that does not require growth and isolation of N. gonorrhoeae in the laboratory. This is problematic because the rates of antibiotic resistance in N. gonorrhoeae are increasing, but without isolating the organism in the clinical laboratory, antibiotic susceptibility testing cannot be performed on strains recovered from clinical specimens. We observed an increase in the frequency of urine cultures growing N. gonorrhoeae after we implemented a total laboratory automation system for culture in our clinical laboratory. Here, we report on the rates of resistance to multiple historically used, first-line, and potential future-use antibiotics for 64 N. gonorrhoeae isolates. We found that the rates of antibiotic resistance in our isolates were comparable to national rates. Additionally, resistance to specific antibiotics correlated closely with the presence of genetic resistance genes, suggesting that DNA-based tests could also be designed to guide antibiotic therapy for treating gonorrhea.

Whole-genome sequencing of Neisseria gonorrhoeae in a forensic transmission case.

Francés-Cuesta C, de la Caba I, Idigoras P, Fernández-Rodríguez A, Del Valle Pérez D, Marimón JM, González-Candelas F. (Full Text)

Forensic Sci Int Genet. 2019 Jul 8;42:141-146. doi: 10.1016/j.fsigen.2019.07.003. [Epub ahead of print] 

Abstract

Molecular epidemiology and phylogenetic analyses are frequently used in the investigation of viral transmission cases in forensic contexts. Here, we present the methods and results of the analysis of a bacterial transmission episode in an alleged child abuse case using complete genome sequences obtained by high-throughput sequencing (HTS) methods.



We obtained genomes of Neisseria gonorrhoeae from the victim, the suspect, and 29 unrelated controls. The analysis of the genomes revealed that the victim and suspect isolates had identical sequences in both the bacterial chromosome and the single plasmid present in them. One of the local controls was very similar (differing in only 2 SNPs) to the case sequences, but the remaining controls were very divergent. Additional cases of identity and very high similarity among controls were observed occasionally, pointing at recent transmission cases.



These results were more discriminative than the previous molecular epidemiology analyses performed at the hospital's Microbiology Service, as Multi-Locus Sequence Typing (MLST) could not distinguish between the suspect/victim and the controls isolates, and Pulse Field Gel Electrophoresis (PFGE) was not able to distinguish between the suspect/victim and one of the local controls.



These results lead us to conclude that complete bacterial genome sequences obtained with HTS technologies may be a valuable tool for establishing recent transmission cases and, although more studies are needed, they have a great potential for being used in forensic analyses.

Use of Whole Genome Sequencing for the Molecular Comparison of Neisseria gonorrhoeae Isolates With Decreased Susceptibility to Extended Spectrum Cephalosporins From 2 Geographically Different Regions in America.

Gianecini RA, Zittermann S, Oviedo C, Galas M, Pardo PR, Allen VG, Galarza P, Melano RG. (Full Text)

Sex Transm Dis. 2019 Aug;46(8):548-555. doi: 10.1097/OLQ.0000000000001011.

Abstract

BACKGROUND:

Neisseria gonorrhoeae isolates with reduced susceptibility or resistance to the recommended first-line antimicrobial therapy have been described in several countries. The purpose of this study was to use genome analyses to compare the molecular characteristics of N. gonorrhoeae isolates with decreased susceptibility to extended-spectrum cephalosporin from Ontario, Canada, and Argentina.



METHODS:

A total of 128 N. gonorrhoeae isolates, collected in 2015, were included. The susceptibility to penicillin G, tetracycline, ciprofloxacin, cefixime, ceftriaxone, and azithromycin was determined using the agar dilution method. Isolates were subjected to whole genome sequencing, and an in silico analysis was performed to identify antimicrobial resistance determinants and for genotyping.



RESULTS:

Decreased susceptibility to extended-spectrum cephalosporin was mainly associated with penA mosaic allele 34.001, together with an mtrR promoter A deletion and porB1b alterations G120K/A121N. N. gonorrhoeae multiantigen sequence typing ST1407 or closely related genotypes were identified circulating in both regions.



CONCLUSIONS:

An international multi-drug resistant clone of N. gonorrhoeae was associated with decreased susceptibility to extended-spectrum cephalosporin (ESC) in 2 different regions in America. Evidence of clonal dissemination of the organism in some regions suggests that the strength of surveillance programs and establishment of collaborative projects are essential.

Emergence and Spread of Neisseria gonorrhoeae Strains with High-level Resistance to Azithromycin in Taiwan from 2001 to 2018.

Liu YH, Wang YH, Liao CH, Hsueh PR. 

Antimicrob Agents Chemother. 2019 Jun 24. pii: AAC.00773-19. doi: 10.1128/AAC.00773-19. [Epub ahead of print] (Full Text)

Abstract

A total of 598 Neisseria gonorrhoeae isolates obtained from patients in Taiwan from 2001 to 2018 were evaluated. The minimum inhibitory concentrations (MICs) of ceftriaxone (CRO) and azithromycin (AZM) against the isolates were determined by the agar dilution method. N. gonorrhoeae isolates with AZM MICs of ≥ 1 μg/ml were identified and characterized by the presence of AZM resistance determinants. For high-level AZM-resistant isolates (AZM-HLR, MIC ≥ 256 μg/ml), genotyping was performed using multilocus sequence typing (MLST) and N. gonorrhoeae multi-antigen sequence typing (NG-MAST). Among the N. gonorrhoeae isolates studied, 8.7% (52/598) exhibited AZM MICs of ≥ 1 μg/ml. Thirteen of the 52 isolates contained A2059G (23S rRNA NG-STAR type1) or C2611T (23S rRNA NG-STAR type 2) mutations. The prevalence of A2059G mutation was higher in AZM-HLR isolates (p < 0.001). The -35A deletion in the promoter region of the mtrR gene did not differ between AZM-HLR isolates (100 %, 10/10) and the isolates with AZM MICs of 1 μg/ml to 64 μg/ml (95.2%, 40/42) (p =1.000). The presence of mutations in the mtrR coding region was significantly different between these two groups, 90% (9/10) and 26.2% (11/42), respectively (p < 0.001). The AZM-HLR isolates, all carrying four mutated A2059G alleles, a -35A deletion, and G45D, were classified as MLST 12039/10899 and NG-MAST 1866/16497. In conclusion, Taiwan is among the countries reporting gonococci with high level resistance to AZM so that single dose of ceftriaxone 1g intramuscularly as the first choice for management of N. gonorrhoeae infection should be evaluated.

 

Novel detection strategies and diagnostics

Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer

Golparian D, Donà V, Sánchez-Busó L, Foerster S, Harris S, Endimiani A, Low N, Unemo M. (Full Text)

Sci Rep. 2018 Dec 4;8(1):17596. doi: 10.1038/s41598-018-35750-4.

Abstract

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is common, compromising gonorrhoea treatment internationally. Rapid characterisation of AMR strains could ensure appropriate and personalised treatment, and support identification and investigation of gonorrhoea outbreaks in nearly real-time. Whole-genome sequencing is ideal for investigation of emergence and dissemination of AMR determinants, predicting AMR, in the gonococcal population and spread of AMR strains in the human population. The novel, rapid and revolutionary long-read sequencer MinION is a small hand-held device that generates bacterial genomes within one day. However, accuracy of MinION reads has been suboptimal for many objectives and the MinION has not been evaluated for gonococci. In this first MinION study for gonococci, we show that MinION-derived sequences analysed with existing open-access, web-based sequence analysis tools are not sufficiently accurate to identify key gonococcal AMR determinants. Nevertheless, using an in house-developed CLC Genomics Workbench including de novo assembly and optimised BLAST algorithms, we show that 2D ONT-derived sequences can be used for accurate prediction of decreased susceptibility or resistance to recommended antimicrobials in gonococcal isolates. We also show that the 2D ONT-derived sequences are useful for rapid phylogenomic-based molecular epidemiological investigations, and, in hybrid assemblies with Illumina sequences, for producing contiguous assemblies and finished reference genomes.

Genotypic determinants of fluoroquinolone and macrolide resistance in Neisseria gonorrhoeae.

Hall CL, Harrison MA, Pond MJ, Chow C, Harding-Esch EM, Sadiq ST. (Full Text)

Sex Health. 2019 Aug 1. doi: 10.1071/SH18225. [Epub ahead of print]

Abstract

BACKGROUND: 

High rates of antimicrobial resistance (AMR) in Neisseria gonorrhoeae hinder effective treatment, but molecular AMR diagnostics may help address the challenge. This study aimed to appraise the literature for resistance-associated genotypic markers linked to fluoroquinolones and macrolides, to identify and review their use in diagnostics. 



METHODS: 

Medline and EMBASE databases were searched and data pooled to evaluate associations between genotype and phenotypic resistance. The minimum inhibitory concentration (MIC) cut-offs were ≤ 0.06 mg L-1 for non-resistance to ciprofloxacin and ≤ 0.5 mg L-1 for non-resistance to azithromycin. 



RESULTS: 

Diagnostic accuracy estimates were limited by data availability and reporting. It was found that: 1) S91 and D95 mutations in the GyrA protein independently predicted ciprofloxacin resistance and, used together, gave 98.6% (95% confidence interval (CI) 98.0-99.0%) sensitivity and 91.4% (95%CI 88.6-93.7%) specificity; 2) the number of 23S rRNA gene alleles with C2611T or A2059G mutations was highly correlated with azithromycin resistance, with mutation in any allele giving a sensitivity and specificity of 66.1% (95%CI 62.1-70.0%) and 98.9% (95%CI 97.5-99.5%) respectively. Estimated negative (NPV) and positive predictive values (PPV) for a 23S rRNA diagnostic were 98.6% (95%CI 96.8-99.4%) and 71.5% (95%CI 68.0-74.8%) respectively; 3) mutation at amino acid position G45 in the MtrR protein independently predicted azithromycin resistance; however, when combined with 23S rRNA, did not improve the PPV or NPV. 



CONCLUSIONS

Viable candidates for markers of resistance detection for incorporation into diagnostics were demonstrated. Such tests may enhance antibiotic stewardship and treatment options.

Improved determination of Neisseria gonorrhoeae gyrase A genotype results in clinical specimens.

Allan-Blitz LT, Ellis OL, Wee R, Truong A, Ebeyan SM, Tan LY, Mokany E, Flynn R, Klausner JD. (Full Text)

J Antimicrob Chemother. 2019 Jul 24. pii: dkz292. doi: 10.1093/jac/dkz292. [Epub ahead of print]

Abstract

BACKGROUND:

The emergence of drug-resistant Neisseria gonorrhoeae has prompted the development of rapid molecular assays designed to determine antimicrobial susceptibility. One common assay uses high-resolution melt analysis to target codon 91 of the gyrase A gene (gyrA) to predict N. gonorrhoeae susceptibility to ciprofloxacin.



METHODS:

We extracted DNA from remnant clinical specimens that had previously tested positive for N. gonorrhoeae using the Aptima Combo 2 for CT/NG assay (Hologic, San Diego, CA, USA). We selected DNA extracts from specimens with indeterminate, WT and mutant gyrA genotype results from a previous study using high-resolution melt analysis to detect the gyrA codon 91 mutation. We re-tested those specimens using the recently CE-marked ResistancePlus GC (beta) assay (SpeeDx, Sydney, Australia).



RESULTS:

Of 86 specimens with indeterminate gyrA genotypes on high-resolution melt analysis, the ResistancePlus GC (beta) assay (SpeeDx) identified 30 (35%) WT, 22 (26%) mutant and 34 (40%) indeterminate gyrA genotypes.



CONCLUSIONS:

The ResistancePlus GC (beta) assay showed improved N. gonorrhoeae gyrA genotype determination compared with a prior gyrA genotypic high-resolution melt assay.

A β-lactamase-producing plasmid from Neisseria gonorrhoeae carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDa TEM-1 penicillinase that hydrolyses ampicillin slowly.

Singh R, Perera SR, Katselis GS, Chumala P, Martin I, Kusalik A, Mitzel KM, Dillon JR. (Full Text)

J Antimicrob Chemother. 2019 Jul 23. pii: dkz306. doi: 10.1093/jac/dkz306. [Epub ahead of print]

Abstract

BACKGROUND:

Seven structurally related β-lactamase-producing plasmids have been characterized in penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates. We characterized a variant (i.e. pJRD20, Canada type) of the Africa-type (pJD5) plasmid isolated from N. gonorrhoeae strain 8903.



OBJECTIVES:

To compare the DNA sequence of pJRD20 with that of pJD5 and pJD4 (Asia-type) and their TEM-1 β-lactamases.



METHODS:

N. gonorrhoeae 8903 was identified as part of the Gonococcal Antimicrobial Surveillance Program in Canada. β-Lactamase production was assessed using nitrocefin. MICs were determined by agar dilution and Etest methods (CLSI). The DNA sequences of pJRD20, pJD5 and pJD4 were assembled and annotated. The structure of TEM-1 and its penicillin-binding properties were determined by in silico molecular modelling and docking. TEM-1 proteins were characterized by western blot, mass spectrometry and ampicillin hydrolysis assays.



RESULTS:

N. gonorrhoeae 8903 exhibited intermediate susceptibility to penicillin with slow β-lactamase activity (i.e. 35 min to hydrolyse nitrocefin). Except for a novel 6 bp deletion starting at the G of the ATG start codon of blaTEM-1, the DNA sequence of pJRD20 was identical to that of pJD5. The TEM-1 β-lactamase produced by pJRD20 is 24 kDa and hydrolyses ampicillin only after several hours.



CONCLUSIONS:

This unusual PPNG isolate might have been characterized as a non-PPNG owing to its low MIC of penicillin and its very slow hydrolysis of nitrocefin. Given the unusual nature of its TEM-1 β-lactamase, laboratories might consider extending the duration of nitrocefin hydrolysis assays.

Using the genetic characteristics of Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to develop a molecular assay to predict cefixime susceptibility.

Deng X, Allan-Blitz LT, Klausner JD. (Full Text)

Sex Health. 2019 Jun 24. doi: 10.1071/SH18227. [Epub ahead of print]

Abstract

BACKGROUND:

In the last two decades, gonococcal strains with decreased cefixime susceptibility and cases of clinical treatment failure have been reported worldwide. Gonococcal strains with a cefixime minimum inhibitory concentration (MIC) ≥0.12 µg mL-1 are significantly more likely to fail cefixime treatment than strains with an MIC <0.12 µg mL-1. Various researchers have described the molecular characteristics of gonococcal strains with reduced cefixime susceptibility, and many have proposed critical molecular alterations that contribute to this decreased susceptibility.



METHODS:

A systematic review of all published articles in PubMed through 1 November 2018 was conducted that report findings on the molecular characteristics and potential mechanisms of resistance for gonococcal strains with decreased cefixime susceptibility. The findings were summarised and suggestions were made for the development of a molecular-based cefixime susceptibility assay.



RESULTS:

The penicillin-binding protein 2 (PBP2) encoded by the penA gene is the primary target of cefixime antimicrobial activity. Decreased cefixime susceptibility is conferred by altered penA genes with mosaic substitute sequences from other Neisseria (N.) species (identifiable by alterations at amino acid position 375-377) or by non-mosaic penA genes with at least one of the critical amino acid substitutions at positions 501, 542 and 551. Based on this review of 415 international cefixime decreased susceptible N. gonorrhoeae isolates, the estimated sensitivity for an assay detecting the aforementioned amino acid alterations would be 99.5% (413/415).



CONCLUSIONS: Targeting mosaic penA and critical amino acid substitutions in non-mosaic penA are necessary and may be sufficient to produce a robust, universal molecular assay to predict cefixime susceptibility.

 

Novel antibiotics, mechanisms, vaccine, and other advances

Commensal Neisseria Kill Neisseria gonorrhoeae through a DNA-Dependent Mechanism.

Kim WJ, Higashi D, Goytia M, Rendón MA, Pilligua-Lucas M, Bronnimann M, McLean JA, Duncan J, Trees D, Jerse AE, So M. (Full Text

Cell Host Microbe. 2019 Jul 31. pii: S1931-3128(19)30347-6. doi: 10.1016/j.chom.2019.07.003. [Epub ahead of print]

Abstract

The mucosa is colonized with commensal Neisseria. Some of these niches are sites of infection for the STD pathogen Neisseria gonorrhoeae (Ngo). Given the antagonistic behavior of commensal bacteria toward their pathogenic relatives, we hypothesized that commensal Neisseria may negatively affect Ngo colonization. Here, we report that commensal species of Neisseria kill Ngo through a mechanism based on genetic competence and DNA methylation state. Specifically, commensal-triggered killing occurs when the pathogen takes up commensal DNA containing a methylation pattern that it does not recognize. Indeed, any DNA will kill Ngo if it can enter the cell, is differentially methylated, and has homology to the pathogen genome. Consistent with these findings, commensal Neisseria elongata accelerates Ngo clearance from the mouse in a DNA-uptake-dependent manner. Collectively, we propose that commensal Neisseria antagonizes Ngo infection through a DNA-mediated mechanism and that DNA is a potential microbicide against this highly drug-resistant pathogen.

The Novel Interaction Between Neisseria gonorrhoeae TdfJ and Human S100A7 Allows Gonococci to Subvert Host Zinc Restriction.

Maurakis S, Keller K, Maxwell CN, Pereira K, Chazin WJ, Criss AK, Cornelissen CN.

PLoS Pathog. 2019 Aug 1;15(8):e1007937. doi: 10.1371/journal.ppat.1007937. [Epub ahead of print]

Abstract

Neisseria gonorrhoeae causes the sexually-transmitted infection gonorrhea, a global disease that is difficult to treat and for which there is no vaccine. This pathogen employs an arsenal of conserved outer membrane proteins called TonB-dependent transporters (TdTs) that allow the gonococcus to overcome nutritional immunity, the host strategy of sequestering essential nutrients away from invading bacteria to handicap infectious ability. N. gonorrhoeae produces eight known TdTs, of which four are utilized for acquisition of iron or iron chelates from host-derived proteins or xenosiderophores produced by other bacteria. Of the remaining TdTs, two of them, TdfH and TdfJ, facilitate zinc uptake. TdfH was recently shown to bind Calprotectin, a member of the S100 protein family, and subsequently extract its zinc, which is then internalized by N. gonorrhoeae. Like Calprotectin, other S100s are also capable of binding transition metals such as zinc and copper, and thus have demonstrated growth suppression of numerous other pathogens via metal sequestration. Considering the functional and structural similarities of the TdTs and of the S100s, as well as the upregulation in response to Zn limitation shown by TdfH and TdfJ, we sought to evaluate whether other S100s have the ability to support gonococcal growth by means of zinc acquisition and to frame this growth in the context of the TdTs. We found that both S100A7 and S10012 are utilized by N. gonorrhoeae as a zinc source in a mechanism that depends on the zinc transport system ZnuABC. Moreover, TdfJ binds directly to S100A7, from which it internalizes zinc. This interaction is restricted to the human version of S100A7, and zinc presence in S100A7 is required to fully support gonococcal growth. These studies highlight how gonococci co-opt human nutritional immunity, by presenting a novel interaction between TdfJ and human S100A7 for overcoming host zinc restriction.

Recognition of the β-lactam Carboxylate Triggers Acylation of Neisseria gonorrhoeae Penicillin-Binding Protein 2.

Singh A, Tomberg J, Nicholas RA, Davies C. (Full Text)

J Biol Chem. 2019 Jul 30. pii: jbc.RA119.009942. doi: 10.1074/jbc.RA119.009942. [Epub ahead of print]

Abstract

Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA gene, encoding penicillin-binding protein 2 (PBP2) variants. Such variants contain up to 62 mutations compared with wild-type, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of wild-type PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr498 sidechain in the KTG motif to contact the cephalosporin carboxylate, twisting of the β3 strand to form the oxyanion hole, and rolling of the β3-β4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of β3 because its sidechain hydroxyl forms a hydrogen bond with Thr498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Prister LL, Ozer EA, Cahoon LA, Seifert HS. (Full Text)

Mol Microbiol. 2019 Jul 24. doi: 10.1111/mmi.14356. [Epub ahead of print]

Abstract

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the type IV pilus. Gene conversion occurs between one of several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation assays (ChIP), we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.

Structural, biochemical and in vivo characterization of MtrR-mediated resistance to innate antimicrobials by the human pathogen Neisseria gonorrhoeae.

Beggs GA, Zalucki YM, Brown NG, Rastegari S, Phillips RK, Palzkill T, Shafer W, Kumaraswami M, Brennan RG. (Full Text)

J Bacteriol. 2019 Jul 22. pii: JB.00401-19. doi: 10.1128/JB.00401-19. [Epub ahead of print]

Abstract 

Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE-encoded multidrug efflux pump, which expels microbicides such as bile salts, fatty acids, and multiple extrinsically administered drugs from the cell. In the absence of these cytotoxins, the TetR-family member, MtrR, represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observe electron density that we hypothesize is cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interaction, both in vitro and in vivo, revealed these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate, but not taurodeoxycholate, suggesting different binding modes of these bile salts. These data provide insight into a crucial mechanism utilized by this pathogen to overcome innate human defences.



IMPORTANCE Neisseria gonorrhoeae causes significant disease burden worldwide and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. Multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage to the pathogen within the host. Transcription of this pump is repressed by MtrR, but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials.

Determination of MIC Quality Control Ranges for the Novel Gyrase Inhibitor, Zoliflodacin.

Miller AA, Traczewski MM, Huband MD, Bradford PA, Mueller JP. (Full Text)

J Clin Microbiol. 2019 Jul 17. pii: JCM.00567-19. doi: 10.1128/JCM.00567-19. [Epub ahead of print]

Abstract

This report describes the results of two different, multi-laboratory QC studies that were used to establish QC ranges for the novel gyrase inhibitor, zoliflodacin, against the ATCC strains recommended by the Clinical and Laboratory Standards Institute (CLSI). Following the completion of an eight-laboratory, CLSI document M23-defined tier 2 study, the agar dilution MIC quality control (QC) range for zoliflodacin against the Neisseria gonorrhoeae QC strain ATCC 49226 was defined as 0.06 - 0.5 μg/mL and was approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing. This QC range will be used for in vitro susceptibility testing of zoliflodacin during phase 3 human clinical trials and surveillance studies, and eventually will be implemented in clinical labs. In a separate study, broth microdilution MIC quality control ranges for zoliflodacin against the following additional QC strains were determined to be: 0.12 - 0.5 μg/mL for Staphylococcus aureus ATCC 29213, 0.25 - 2 μg/mL for Enterococcus faecalis ATCC 29212, 1 - 4 μg/mL for Escherichia coli ATCC 25922, 0.12 - 0.5 μg/mL for Streptococcus pneumoniae ATCC 49619 and 0.12 - 1 μg/mL for Haemophilus influenzae ATCC 49247. These MIC QC ranges were also approved by CLSI for use in future in vitro susceptibility testing studies against organisms other than N. gonorrhoeae.

New treatment options for Neisseria gonorrhoeae in the era of emerging antimicrobial resistance.

Lewis DA. (Full Text)

Sex Health. 2019 Jul 11. doi: 10.1071/SH19034. [Epub ahead of print]

Abstract

Neisseria gonorrhoeae, the causative agent of gonorrhoea, has rapidly evolved from an exquisitely susceptible pathogen into a 'superbug' with the capacity to exhibit an extensively drug resistant (XDR) phenotype. The threat of untreatable gonorrhoea now looms on the horizon while the arsenal of effective antimicrobial agents diminishes with time. Ceftriaxone remains the mainstay of first-line therapy as a single agent or as the backbone of a dual therapy regimen. The implementation of new assays to facilitate 'precision' treatment, based on the prediction of N. gonorrhoeae susceptibility to old anti-gonococcal drugs, may enable sparing use of ceftriaxone in those countries that can afford this technology. A few existing drugs, such as ertapenem, can be repositioned to help manage multi-drug resistant and XDR gonorrhoea. Recent clinical trials involving solithromycin and delafloxacin have generated disappointing results in that both agents failed to show non-inferiority to conventional ceftriaxone-based regimens. At present, zoliflodacin and gepotidacin appear to be the most promising antimicrobial agents in clinical development. Both drugs performed well in eradicating urogenital gonorrhoea in recent Phase 2 trials; however, treatment failures were reported at the oropharyngeal site, which is an important site of infection in men who have sex with men and sex workers. Given this observation, it is unlikely that either of these new agents could be promoted for monotherapy of gonorrhoea. The pre-clinical pipeline remains relatively empty of agents likely to progress to clinical development for gonorrhoea treatment and increased investment into gonorrhoea-specific drug discovery is recommended.

Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells.

Semchenko EA, Everest-Dass AV, Jen FE, Mubaiwa TD, Day CJ, Seib KL. (Full Text)

MBio. 2019 Jul 9;10(4). pii: e01339-19. doi: 10.1128/mBio.01339-19.

Abstract

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 μM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeae



IMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.