Gonorrhoeae Strains Research Articles

Neisseria gonorrhoeae-derived outer membrane vesicles package β-lactamases to promote antibiotic resistance.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea. The treatment of gonorrhoea is becoming increasingly challenging, as N. gonorrhoeae has developed resistance to antimicrobial agents routinely used in the clinic. Resistance to penicillin is wide-spread partly due to the acquisition of β-lactamase genes. How N. gonorrhoeae survives an initial exposure to β-lactams before acquiring resistance genes remains to be understood. Here, using a panel of clinical isolates of N. gonorrhoeae we show that the β-lactamase enzyme is packaged into outer membrane vesicles (OMVs) by strains expressing blaTEM-1B or blaTEM-106, which protects otherwise susceptible clinical isolates from the β-lactam drug amoxycillin. We characterized the phenotypes of these clinical isolates of N. gonorrhoeae and the time courses over which the cross-protection of the strains is effective. Imaging and biochemical assays suggest that OMVs promote the transfer of proteins and lipids between bacteria. Thus, N. gonorrhoeae strains secret antibiotic degrading enzymes via OMVs enabling survival of otherwise susceptible bacteria.

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance.

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC.

In vitro Susceptibility to β-Lactam Antibiotics and Viability of Neisseria gonorrhoeae Strains Producing Plasmid-Mediated Broad- and Extended-Spectrum β-Lactamases.

Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance. The emergence of clinical isolates producing plasmid β-lactamases that can hydrolyze cephalosporins, the mainstay treatment for gonorrhea, may be a serious threat. In this work, N. gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum β-lactamases (ESBLs) were obtained in vitro, and their viability and β-lactam antibiotic susceptibility were studied. Artificial pblaTEM-1 and pblaTEM-20 plasmids were constructed by site-directed mutagenesis from a pblaTEM-135 plasmid isolated from a clinical isolate. Minimum inhibitory concentration (MIC) values for a series of β-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem, were determined. The N. gonorrhoeae strain carrying the pblaTEM-20 plasmid exhibited a high level of resistance to penicillins and second–fourth-generation cephalosporins (MIC ≥2 mg/L) but not to carbapenems (MIC ≤0.008 mg/L). However, this strain stopped growing after 6 h of culture. The reduction in viability was not associated with loss of the plasmid but can be explained by the presence of the plasmid itself, which requires additional reproduction costs, and to the expression of ESBLs, which can affect the structure of the peptidoglycan layer in the cell membrane. Cell growth was mathematically modeled using the generalized Verhulst equation, and the reduced viability of the plasmid-carrying strains compared to the non-plasmid-carrying strains was confirmed. The cell death kinetics of N. gonorrhoeae strains without the pblaTEM-20 plasmid in the presence of ceftriaxone can be described by a modified Chick–Watson law. The corresponding kinetics of the N. gonorrhoeae strain carrying the pblaTEM-20 plasmid reflected several processes: the hydrolysis of ceftriaxone by the TEM-20 β-lactamase and the growth and gradual death of cells. The demonstrated reduction in the viability of N. gonorrhoeae strains carrying the pblaTEM-20 plasmid probably explains the absence of clinical isolates of ESBL-producing N. gonorrhoeae.

Immuno-proteomics of sera from gonorrhoea patients identified potential vaccine candidates

Gonorrhea is a sexually transmitted disease caused by Neisseria gonorrhoeae and is one of the leading reportable STDs in adults, with ~87 million cases annually and globally. Gonorrhoea remains a major global public health concern not only because of rising incidence each year, but also because of rising antimicrobial resistance. There is an urgent need for long-term solutions to prevent gonorrhoea such as vaccines, but none currently existand research is focused on identifying potential antigens for inclusion in new vaccines. In our study, we used an immuno-proteomics approach to try and identify potential vaccine candidates. A heterologous N. gonorrhoeae strain P9-17 was grown under iron-limiting conditions and whole cell lysates prepared. These were separated by isoelectric focusing (pH 3-10 range) and fractions were separated by SDS-PAGE. Western blots were prepared and reacted with sera from 20 patients with uncomplicated gonorrhoea and with sera from 5 controls with no history of gonorrhoea. Immuno-reactive bands were excised from the corresponding gels and subjected to mass spectrometry, which provided a profile of gonococcal proteins. After comparing patterns of reactivity, we identified 180 bands in sera from gonorrhoea patients. Using a bio-informatics approach we refined the list to 18 bands of interest and identified 13 novel proteins associated with an increase in immuno-reactivity with gonococci. In summary, we have identified a unique set of gonococcal proteins that have not yet been investigated as potential vaccine antigens and that are the focus of current studies to develop a gonococcal vaccine.

The European response to control and manage multi- and extensively drug-resistant Neisseria gonorrhoeae.

Because cefixime and ceftriaxone resistance in Neisseria gonorrhoeae and gonorrhoea treatment failures were increasing, a response plan to control and manage multidrug-resistant N. gonorrhoeae (MDR-NG) in Europe was published in 2012. The three main areas of the plan were to: (i) strengthen surveillance of antimicrobial resistance (AMR), (ii) implement monitoring of treatment failures and (iii) establish a communication strategy to increase awareness and disseminate AMR results. Since 2012, several additional extensively drug-resistant N. gonorrhoeae (XDR-NG) strains have emerged, and strains with high-level ceftriaxone resistance spread internationally. This prompted an evaluation and review of the 2012 European Centre for Disease Prevention and Control (ECDC) response plan, revealing an overall improvement in many aspects of monitoring AMR in N. gonorrhoeae; however, treatment failure monitoring was a weakness. Accordingly, the plan was updated in 2019 to further support European Union/European Economic Area (EU/EEA) countries in controlling and managing the threat of MDR/XDR-NG in Europe through further strengthening of AMR surveillance and clinical management including treatment failure monitoring. The plan will be assessed biennially to ensure its effectiveness and its value. Along with prevention, diagnostic, treatment and epidemiological surveillance strategies, AMR surveillance is essential for effective control of gonorrhoea.

The Effect of Efflux Pumps and Oxidative Stress on Neisseria gonorrhoeae Susceptibility to Resazomycins

The increasing prevalence of multidrug-resistant Neisseria gonorrhoeae strains underscores the need for novel antimicrobials against this pathogen. Resazomycins, derivatives of resazurin (Rz), have shown robust antimicrobial activity against N. gonorrhoeae (Ng) in vitro. In vivo, however, resazomycins exhibit limited efficacy in a mouse model of gonorrhea. Previous experiments have shown that N. gonorrhoeae is more resistant to resazurin at oxygen levels comparable to those seen in host tissue (2%). We hypothesized this difference in susceptibility at low oxygen compared to atmospheric oxygen (~20%) was due to altered activity of multi-drug efflux pumps. To test this, we screened a selection of N. gonorrhoeae mutants that do not express or overexpress either the MtrCDE or NorM efflux pumps for Rz susceptibility. Overexpression of MtrCDE resulted in increased resistance to Rz at both 2% and ~20% oxygen suggesting resazurin may be a substrate of this efflux pump. Loss of expression of either MtrCDE or NorM had no effect on the increased resistance of N. gonorrhoeae to resazurin at low oxygen. We next sought to determine whether the increased susceptibility of Rz at 20% oxygen is due to oxidative stress. To test this, we measured the susceptibility of N. gonorrhoeae to Rz in the presence and absence of the antioxidants, cysteine HCl and glutathione, at 20% oxygen. In the presence of cysteine HCl or glutathione, multiple N. gonorrhoeae strains had a higher Rz MIC at 20% oxygen. Here, we have shown oxygen concentration affects N. gonorrhoeae susceptibility to Rz due to increased oxidative stress.

Pharmacodynamic Evaluation of Zoliflodacin Treatment of Neisseria gonorrhoeae Strains With Amino Acid Substitutions in the Zoliflodacin Target GyrB Using a Dynamic Hollow Fiber Infection Model.

Novel antimicrobials for effective treatment of uncomplicated gonorrhea are essential, and the first-in-class, oral spiropyrimidinetrione DNA gyrase B inhibitor zoliflodacin appears promising. Using our newly developed Hollow Fiber Infection Model (HFIM), the pharmacodynamics of zoliflodacin was examined. A clinical zoliflodacin-susceptible N. gonorrhoeae strain, SE600/18 (harbouring a GyrB S467N amino acid substitution; MIC = 0.25 mg/L), and SE600/18-D429N (zoliflodacin-resistant mutant with a second GyrB substitution, D429N, selected in the HFIM experiments; zoliflodacin MIC = 2 mg/L), were examined. Dose-range experiments, simulating zoliflodacin single oral dose regimens of 0.5, 1, 2, 3, and 4 g, were performed for SE600/18. For SE600/18-D429N, dose-range experiments, simulating zoliflodacin single oral 2, 3, 4, and 6 g doses, and zoliflodacin oral dose-fractionation experiments with 4, 6, and 8 g administered as q12 h were performed. Both strains grew well in the untreated HFIM growth control arms and mostly maintained growth at 1010–1011 CFU/ml for 7 days. Zoliflodacin 3 and 4 g single dose oral regimens successfully eradicated SE600/18 and no growth was recovered during the 7-days experiments. However, the single oral 0.5, 1, and 2 g doses failed to eradicate SE600/18, and zoliflodacin-resistant populations with a GyrB D429N substitution were selected with all these doses. The zoliflodacin-resistant SE600/18-D429N mutant was not eradicated with any examined treatment regimen. However, this in vitro-selected zoliflodacin-resistant mutant was substantially less fit compared to the zoliflodacin-susceptible SE600/18 parent strain. In conclusion, the rare clinical gonococcal strains with GyrB S467N substitution are predisposed to develop zoliflodacin resistance and may require treatment with zoliflodacin ≥3 g. Future development may need to consider the inclusion of diagnostics directed at identifying strains resistant or predisposed to resistance development at a population level and to strengthen surveillance (phenotypically and genetically), and possibly also at the patient level to guide treatment.

First characterisation of antimicrobial susceptibility and resistance of Neisseria gonorrhoeae isolates in Qatar, 2017–2020

Limited data are available regarding antimicrobial resistance in Neisseria gonorrhoeae strains circulating in WHO Eastern Mediterranean Region (EMR). We investigated the antimicrobial susceptibility/resistance of N. gonorrhoeae isolates to five antimicrobials (ceftriaxone, azithromycin, ciprofloxacin, tetracycline, and benzylpenicillin) currently or previously used for gonorrhoea treatment in Qatar, 2017–2020. Minimum inhibitory concentrations (MICs; mg/L) of antimicrobials were determined using Etest on gonococcal isolates collected during January 1, 2017-August 30, 2020 at Hamad Medical Corporation, a national public healthcare provider. During 2017–2020, resistance in isolates from urogenital sites of 433 patients was 64.7% (95% CI: 59.5–69.6%; range: 43.9–78.7%) for ciprofloxacin, 50.7% (95% CI: 45.3–56.1%; range: 41.3–70.4%) for tetracycline, and 30.8% (95% CI: 26.3–35.6%; range: 26.7–35.8%) for benzylpenicillin. Percentage of isolates non-susceptible to azithromycin was 4.1% (95% CI: 2.0–7.4%; range: 2.7–4.8%) and all (100%) isolates were susceptible to ceftriaxone. Two (1.6%) isolates from 2019 and one (2.2%) isolate from 2020 had high-level resistance to azithromycin (MIC≥256 mg/L). Overall, 1.0% (4/418) of isolates had a ceftriaxone MIC of 0.25 mg/L, which is at the ceftriaxone susceptibility breakpoint (MIC≤0.25 mg/L). Treatment with ceftriaxone 250 mg plus azithromycin 1 g can continuously be recommended for gonorrhoea therapy in Qatar. Continued quality-assured gonococcal AMR surveillance is warranted in EMR.

Evaluation of Neisseria gonorrhoeae Isolates Susceptibility to Tetracycline Antibiotics from 9 Provinces in China Since 2020.

PurposeThe increasing drug resistance of Neisseria gonorrhoeae has become a serious public health concern. This study investigated N. gonorrhoeae isolates susceptibility to tetracycline antibiotics and the correlation between minimum inhibitory concentrations (MICs) of different antibiotics. The presence of resistance determinants in N. gonorrhoeae strains displaying different levels of tigecycline resistance was also compared.MethodsThe minimum inhibitory concentrations (MICs) of tetracycline, minocycline, tigecycline, eravacycline, omadacycline on 412 N. gonorrhoeae isolates were measured by the agar dilution method. The MICs of ceftriaxone and azithromycin were also measured to determine the correlations between antibiotics by the value of the correlation coefficient R. The presence of resistance determinants was identified through polymerase chain reaction (PCR) and sequencing.ResultsThe MIC90 was 64mg/L for tetracycline, 64mg/L for minocycline, 0.5mg/L for tigecycline, 0.5mg/L for eravacycline, 4mg/L for omadacycline, 0.25 mg/L for ceftriaxone, and 1mg/L for azithromycin. The MIC90 and mode of tigecycline and eravacycline were much lower than those of tetracycline and minocycline. A poor correlation between omadacycline, eravacycline and tetracycline susceptibility was observed. Minocycline has a strong correlation with tetracycline. PorB1 typing, TetM-encoding plasmid, and mtrR promoter adenine deletion were significantly correlated with tigecycline MIC > 0.25mg/L.ConclusionThis study suggested that tigecycline and eravacycline had better in vitro activity and might be alternative antibiotics against resistant N. gonorrhoeae infections. Nevertheless, further in vitro experiments and clinical studies are needed for verification.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™2×300bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

Pharmacodynamics of Ceftriaxone, Ertapenem, Fosfomycin and Gentamicin in Neisseria gonorrhoeae.

Objectives: To assess the in vitro effect of select antimicrobials on the growth of N. gonorrhoeae and its pharmacodynamic parameters. Methods: Time–kill assays were performed on two reference N. gonorrhoeae strains (ceftriaxone-resistant WHO X and ceftriaxone-susceptible WHO F) and one clinical N. gonorrhoeae strain (ceftriaxone-susceptible CS03307). Time–kill curves were constructed for each strain by measuring bacterial growth rates at doubling antimicrobial concentrations of ceftriaxone, ertapenem, fosfomycin and gentamicin. Inputs from these curves were used to estimate minimal bacterial growth rates at high antimicrobial concentrations (ψmin), maximum bacterial growth rates in the absence of antimicrobials (ψmax), pharmacodynamic minimum inhibitory concentrations (zMIC), and Hill’s coefficients (κ). Results: Ceftriaxone, ertapenem and fosfomycin showed gradual death overtime at higher antimicrobial concentrations with a relatively high ψmin, demonstrating time-dependent activity. Compared to WHO F, the ψmin for WHO X was significantly increased, reflecting decreased killing activity for ceftriaxone, ertapenem and fosfomycin. At high ceftriaxone concentrations, WHO X was still efficiently killed. CS03307 also showed a high ψmin for ceftriaxone in spite of a low MIC and no difference in ψmin for fosfomycin in spite of significant MIC and zMIC differences. Gentamicin showed rapid killing for all three strains at high concentrations, demonstrating concentration-dependent activity. Conclusions: Based on time–kill assays, high-dosage ceftriaxone could be used to treat N. gonorrhoeae strains with MIC above breakpoint, with gentamicin as a potential alternative. Whether ertapenem or fosfomycin would be effective to treat strains with a high MIC to ceftriaxone is questionable.

Dissemination and genome analysis of high-level ceftriaxone-resistant penA 60.001 Neisseria gonorrhoeae strains from the Guangdong Gonococcal antibiotics susceptibility Programme (GD-GASP), 2016–2019

ABSTRACT Background: After Neisseria gonorrhoeae FC428 was first found in Japan, ceftriaxone-resistant strains disseminated globally, and the gonococcal resistance rate increased remarkably. Epidemiological investigations are greatly significant for the analysis of antimicrobial resistance (AMR) trends, molecular features and evolution. Objectives: To clarify the AMR trend from 2016–2019 and reveal the molecular characteristics and evolution of ceftriaxone-resistant penA 60.001 isolates. Methods: The minimum inhibitory concentrations (MICs) of antibiotics against 4113 isolates were detected by the agar dilution method. N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST) and N.gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were used to identify the sequence types. Genome analysis was conducted to analyze resistance genes, virulence factors, and evolutionary sources. Results: Isolates with decreased ceftriaxone susceptibility have increased from 2.05% (2016) to 16.18% (2019). Six ceftriaxone-resistant isolates possessing penA 60.001 appeared in Guangdong Province, and were resistant to ceftriaxone, penicillin, tetracycline, ciprofloxacin and cefixime, but susceptible to azithromycin and spectinomycin. Single-nucleotide polymorphisms (SNPs) in the porB gene were the major cause of different NG-MAST types. ST1903 was the main NG-STAR genotype and only strain-ZH545 was ST7365, with molecular features consistent with the MICs. Furthermore, different MLSTs suggested diverse evolutionary sources. Genome analysis revealed a set of virulence factors along with the resistance genes “penA” and “blaTEM-1B”. Half of penA 60.001 strains were fully mixed with global FC428-related strains. Conclusions: Global FC428-related clones have disseminated across Guangdong, possibly causing decreased ceftriaxone susceptibility. Enhanced gonococcal surveillance will help elucidate the trajectory of transmission and curb further dissemination.

РЕЗУЛЬТАТЫ МОНИТОРИНГА АНТИБАКТЕРИАЛЬНОЙ РЕЗИСТЕНТНОСТИ N. GONORRHOEAE В г. МОГИЛЕВЕ ЗА 2017-2019 г.

The article presents the results of a clinical study conducted in 2018 and 2020 to determine the antibiotic resistance indicators for N. gonorrhoea strains in Belarus. One hundred and fourteen (114) cultures of N. gonorrhoeae from Mogilev were included in the works: thirty six (36) cultures were isolated from patients in 2017, nineteen (19) in 2018 and fifty nine (59) in 2019. All samples were obtained from the cervical canal in women or the urethra in men, with an established diagnosis of gonorrhea. Men concluded 82 persons equal to 72 %, and women 32 equal 28 %. The average age of men was 23.5 (range: 17—44 years), women 23,5 (range: 17—74 years). The susceptibility to ceftriaxone, cefixime, azithromycin, ciprofloxacin, spectinomycin, benzylpenicillin, tetracycline and gentamicin were determined using the Etest for determination of the minimum inhibitory concentrations. Recommendations on the use of certain antimicrobial agents in the treatment of gonorrhea is based on data obtained.

Emerging Azithromycin Resistance among the Neisseria gonorrhoeae Strains Isolated in Danyang, China

Emerging Azithromycin Resistance among the Neisseria gonorrhoeae Strains Isolated in Danyang, China

Modelling response strategies for controlling gonorrhoea outbreaks in men who have sex with men in Australia.

The ability to treat gonorrhoea with current first-line drugs is threatened by the global spread of extensively drug resistant (XDR) Neisseria gonorrhoeae (NG) strains. In Australia, urban transmission is high among men who have sex with men (MSM) and importation of an XDR NG strain in this population could result in an epidemic that would be difficult and costly to control. An individual-based, anatomical site-specific mathematical model of NG transmission among Australian MSM was developed and used to evaluate the potential for elimination of an imported NG strain under a range of case-based and population-based test-and-treat strategies. When initiated upon detection of the imported strain, these strategies enhance the probability of elimination and reduce the outbreak size compared with current practice (current testing levels and no contact tracing). The most effective strategies combine testing targeted at regular and casual partners with increased rates of population testing. However, even with the most effective strategies, outbreaks can persist for up to 2 years post-detection. Our simulations suggest that local elimination of imported NG strains can be achieved with high probability using combined case-based and population-based test-and-treat strategies. These strategies may be an effective means of preserving current treatments in the event of wider XDR NG emergence.

Self-Inhibitory Peptides Targeting the Neisseria gonorrhoeae MtrCDE Efflux Pump Increase Antibiotic Susceptibility.

ABSTRACTNeisseria gonorrhoeae is an increasing public health threat due to its rapidly rising incidence and antibiotic resistance. There are an estimated 106 million cases per year worldwide, there is no vaccine available to prevent infection, and N. gonorrhoeae strains that are resistant to all antibiotics routinely used to treat the infection have emerged. In many strains, antibiotic resistance is mediated by overexpression of the MtrCDE efflux pump, which enables the bacteria to transport toxic antibiotics out of the cell. Genetic mutations that inactivate MtrCDE have previously been shown to render resistant strains susceptible to certain antibiotics. Here, we show that peptides rationally designed to target and disrupt the activity of each of the three protein components of MtrCDE were able to increase the susceptibility of N. gonorrhoeae strains to antibiotics in a dose-dependent manner and with no toxicity to human cells. Cotreatment of bacteria with subinhibitory concentrations of the peptide led to 2- to 64-fold increases in susceptibility to erythromycin, azithromycin, ciprofloxacin, and/or ceftriaxone in N. gonorrhoeae strains FA1090, WHO K, WHO P, and WHO X. The cotreatment experiments with peptides P-MtrC1 and P-MtrE1 resulted in increased susceptibilities of WHO P and WHO X to azithromycin, ciprofloxacin, and ceftriaxone that were of the same magnitude seen in MtrCDE mutants. P-MtrE1 was able to change the azithromycin resistance profile of WHO P from resistant to susceptible. Data presented here demonstrate that these peptides may be developed for use as a dual treatment with existing antibiotics to treat multidrug-resistant gonococcal infections.

Combating Antimicrobial Resistance: Assessing Efflux Pump Deficient Environments in Neisseria gonorrhoeae and the Masking Effect on Antimicrobial-Resistant Mutations

Introduction: Antimicrobial resistance (AMR) is a global emergency that involves the process of microbes gaining resistance to antimicrobials. A specific example of this is Neisseria (N.) gonorrhoeae gaining resistance to azithromycin, which can be caused by mutations at ribosomal sites. New research suggests that manipulating the bacteria to create a drug efflux pump deficiency will “mask” the effects of AMR mutations when introduced to antibiotic drugs, compared to that of a drug efflux pump proficient environment. This proposal aims to investigate the effects of a drug efflux pump deficient environment compared to a proficient environment on azithromycin drug efficacy in N. gonorrhoeae with an AMR ribosomal mutation. It is hypothesized that AMR mutations can be masked through gene knockout causing MtrCDE efflux pump deficient environments of N. gonorrhoeae. Methods: Aim 1 consists of inducing a ribosomal mutation that incurs resistance to azithromycin in N. gonorrhoeae strains using CRISPR genome editing in groups A and B. Aim 2 will utilize CRISPR-Cas9 to delete the mtrE gene in N. gonorrhoeae, rendering the MtrCDE efflux pump deficient, creating an efflux pump deficient environment in the bacteria of groups B and D. Aim 3 will determine the difference in susceptibility of N. gonorrhoeae towards azithromycin between efflux pump deficient and proficient environments, using disk diffusion and zone of inhibition. Results: We expect Group A will have a smaller diameter zone of inhibition exhibiting azithromycin resistance, while Groups B, C and D will result in a larger diameter exhibiting azithromycin susceptibility. Discussion: We anticipate that Group A will have the smallest diameter zone of inhibition due to the azithromycin resistant mutation and proficient efflux pump environment. We expect Group B will have the largest diameter, demonstrating that N. gonorrhoeae became susceptible to azithromycin with the deficient efflux pump environment masking the azithromycin resistant mutation. Group C (proficient pump) and D (deficient pump) are both wild-type strains therefore we predict they will have larger diameters due to their natural susceptibility to azithromycin. Conclusion: The proposed experiment could help uncover the molecular mechanisms behind masking, which could consequently provide a gateway towards effective strategies to combat AMR.

A drug candidate for Alzheimer's and Huntington's disease, PBT2, can be repurposed to render Neisseria gonorrhoeae susceptible to natural cationic antimicrobial peptides.

Neisseria gonorrhoeae is a Gram-negative bacterial pathogen that causes gonorrhoea. No vaccine is available to prevent gonorrhoea and the emergence of MDR N. gonorrhoeae strains represents an immediate public health threat. To evaluate whether PBT2/zinc may sensitize MDR N. gonorrhoeae to natural cationic antimicrobial peptides. MDR strains that contain differing resistance mechanisms against numerous antibiotics were tested in MIC assays. MIC assays were performed using the broth microdilution method according to CLSI guidelines in a microtitre plate. Serially diluted LL-37 or PG-1 was tested in combination with a sub-inhibitory concentration of PBT2/zinc. Serially diluted tetracycline was also tested with sub-inhibitory concentrations of PBT2/zinc and LL-37. SWATH-MS proteomic analysis of N. gonorrhoeae treated with PBT2/zinc, LL-37 and/or tetracycline was performed to determine the mechanism(s) of N. gonorrhoeae susceptibility to antibiotics and peptides. Sub-inhibitory concentrations of LL-37 and PBT2/zinc synergized to render strain WHO-Z susceptible to tetracycline, whereas the killing effect of PG-1 and PBT2/zinc was additive. SWATH-MS proteomic analysis suggested that PBT2/zinc most likely leads to a loss of membrane integrity and increased protein misfolding and, in turn, results in bacterial death. Here we show that PBT2, a candidate Alzheimer's and Huntington's disease drug, can be repurposed to render MDR N. gonorrhoeae more susceptible to the endogenous antimicrobial peptides LL-37 and PG-1. In the presence of LL-37, PBT2/zinc can synergize with tetracycline to restore tetracycline susceptibility to gonococci resistant to this antibiotic.

In vitro evaluation of antimicrobial resistance selection in Neisseria gonorrhoeae

Gonococcal infections represent an urgent public-health threat as >50% of cases caused by Neisseria gonorrhoeae strains display reduced susceptibility to at least one antimicrobial agent. We evaluated the pharmacodynamics of a number of antimicrobials against N. gonorrhoeae in order to assess the likelihood of mutant selection by these agents. The mutant prevention concentration (MPC) and mutant selection window (MSW) were determined for azithromycin, ceftriaxone, doxycycline, ertapenem, gentamicin, ciprofloxacin, levofloxacin and moxifloxacin against a wild-type strain of N. gonorrhoeae (ATCC 49226) and a gyrA mutant of ATCC 49226. Pharmacokinetic parameters, including peak concentration (Cmax), half-life (t1/2) and area under the plasma concentration-time curve over 24 h (AUC), associated with each agent were used to calculate the time within the MSW (TMSW, percentage of the dosing interval that antimicrobial concentrations fall within the MSW), Cmax/MPC ratio and AUC/MPC ratio for each antimicrobial agent. Concentrations of ceftriaxone (500 mg), ertapenem, ciprofloxacin, levofloxacin and moxifloxacin surpass the MPC for both strains. Results of pharmacodynamic analyses suggest that ertapenem, ciprofloxacin, levofloxacin and moxifloxacin may be most likely to prevent mutant selection in N. gonorrhoeae. Use of ceftriaxone, azithromycin, doxycycline or gentamicin for gonorrhoea is expected to lead to the ongoing emergence of resistance to these agents. There is a clear need to develop novel treatment regimens for gonococcal infections in order to limit the dissemination of resistance in N. gonorrhoeae.

Molecular characterization of a ceftriaxone-resistant Neisseria gonorrhoeae strain found in Switzerland: a case report

BackgroundThe resistance of Neisseria gonorrhoeae to ceftriaxone is unusual in Switzerland. The underlying genotype responsible for resistance is suspected to be novel. Generally, resistance in Neisseria gonorrhoeae (Ng) involves a comprehensive set of genes with many different mutations leading to resistance to different β-lactams and fluoroquinolones.Case presentationA patient had a positive result from specific PCR for Ng. We routinely culture all clinical specimens with a positive NG-PCR. In this particular case, we isolated a strain with resistance to ceftriaxone in Switzerland. A total of seven different genes (penA, ponA, porinB, mtr, gyrA, parC, 23S rRNA gene) in this strain were partially sequenced for comparison with phenotypic susceptibility testing. Interestingly, two different mutations in the porinB gene were observed, and data on this gene are limited. Information on the identified allele type of the penA gene is very limited as well. Three different mutations of parC and gyrA that correlate with ciprofloxacin resistance were found. The combination of ceftriaxone and ciprofloxacin resistance makes an appropriate treatment difficult to obtain due to multidrug resistance.ConclusionThe combined results for all genes show the appearance of new mutations in central Europe either due to worldwide spread or the emergence of new genetic combinations of mutations.