Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

A Single Amino Acid Substitution in Elongation Factor G Can Confer Low-Level Gentamicin Resistance in Neisseria gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.

Could Dampening Expression of the Neisseria gonorrhoeae mtrCDE-Encoded Efflux Pump Be a Strategy To Preserve Currently or Resurrect Formerly Used Antibiotics To Treat Gonorrhea?

Neisseria gonorrhoeae has developed resistance to every antibiotic introduced for treatment of gonorrhea since 1938, and concern now exists that gonorrheal infections may become refractory to all available antibiotics approved for therapy. The current recommended dual antibiotic treatment regimen of ceftriaxone (CRO) and azithromycin (AZM) is threatened with the emergence of gonococcal strains displaying resistance to one or both of these antibiotics. Non-beta-lactamase resistance to penicillin and third-generation cephalosporins, as well as low-level AZM resistance expressed by gonococci, requires overexpression of the mtrCDE-encoded efflux pump, which in wild-type (WT) strains is subject to transcriptional repression by MtrR. Since earlier studies showed that loss of MtrCDE renders gonococci hypersusceptible to beta-lactams and macrolides, we hypothesized that transcriptional dampening of mtrCDE would render an otherwise resistant strain susceptible to these antibiotics as assessed by antibiotic susceptibility testing and during experimental infection. In order to test this hypothesis, we ectopically expressed a WT copy of the mtrR gene, which encodes the repressor of the mtrCDE efflux pump operon, in N. gonorrhoeae strain H041, the first reported gonococcal strain to cause a third-generation-cephalosporin-resistant infection. We now report that MtrR production can repress the expression of mtrCDE, increase antimicrobial susceptibility in vitro, and enhance beta-lactam efficacy in eliminating gonococci as assessed in a female mouse model of lower genital tract infection. We propose that strategies that target the MtrCDE efflux pump should be considered to counteract the increasing problem of antibiotic-resistant gonococci.IMPORTANCE The emergence of gonococcal strains resistant to past or currently used antibiotics is a global public health concern, given the estimated 78 million infections that occur annually. The dearth of new antibiotics to treat gonorrhea demands that alternative curative strategies be considered to counteract antibiotic resistance expressed by gonococci. Herein, we show that decreased expression of a drug efflux pump that participates in gonococcal resistance to antibiotics can increase gonococcal susceptibility to beta-lactams and macrolides under laboratory conditions, as well as improve antibiotic-mediated clearance of gonococci from the genital tract of experimentally infected female mice.

Evidence of Recent Genomic Evolution in Gonococcal Strains With Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014-2016.

BACKGROUND: Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States, 2014-2016. METHODS: We sequenced genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project. We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations. RESULTS: We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin minimum inhibitory concentration (AZIem), characterized by a mosaic mtr locus (C substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZIem strains. Another clonal group (MLST ST1901) possessed 7 unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance. CONCLUSIONS: Whole-genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection in a patient.

Copper Ions and Coordination Complexes as Novel Carbapenem Adjuvants.

Carbapenem-resistant Enterobacteriaceae are urgent threats to global human health. These organisms produce ß-lactamases with carbapenemase activity, such as the metallo-ß-lactamase NDM-1, which is notable due to its association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive Escherichia coli isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse ß-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other ß-lactamases, the blaNDM-1 gene from MS6192 was cloned and expressed in a recombinant E. coli K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 µM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.

Antimicrobial Resistance Expressed by Neisseria gonorrhoeae: A Major Global Public Health Problem in the 21st Century.

Neisseria gonorrhoeae is a strictly human pathogen that is typically transmitted by sexual contact. The associated disease gonorrhea has plagued humankind for thousands of years, with a current estimated incidence of 78 million cases per year. Advances in antimicrobial discovery in the 1920s and 1930s leading to the discovery of sulfonamides and penicillin begun the era of effective antimicrobial treatment of gonorrhea. Unfortunately, the gonococcus developed decreased susceptibility or even resistance to these initially employed antibiotics, a trend that continued over subsequent decades with each new antibiotic that was brought into clinical practice. As this pattern of resistance has continued into the 21st century, there is now reason for great concern, especially in an era when few new antibiotics have prospects for use as treatment of gonorrhea. Here, we review the history of gonorrhea treatment regimens and gonococcal resistance to antibiotics, the mechanisms of resistance, resistance monitoring schemes that exist in different international settings, global responses to the challenge of resistance, and prospects for future treatment regimens in the 21st century.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.