Yasmin Surani: Convergent evolution of antibiotic resistance mechanisms between pyrrolobenzodiazepines and albicidin in multidrug resistant Klebsiella pneumoniae

Exposure to antibiotics presents a survival challenge to the bacteria, which may result in the emergence of resistant populations through genomic mutations followed by either vertical or horizontal transmission2. Multidrug-resistant (MDR) bacterial populations are on the rise, and pose a real threat to public health, both economically and with regards to the loss of human life1.

Species of particular concern have been termed the ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species3. Of these, the last four are Gram-negative bacteria, which present a greater challenge due to the need to penetrate their two cell membranes with very different properties while avoiding drug efflux4,5.

K. pneumoniae inhabits the gastrointestinal tract, but it is also an opportunistic hospital-associated pathogen. One-third of all Gram-negative infections are attributed to it, including pneumonia, urinary tract infections, wound colonisation, and, more seriously: endocarditis, and septicaemia. K. pneumoniae strains have exhibited resistance to the four major antibiotic classes: third generation cephalosporins, aminoglycosides, fluoroquinolones, and even carbapenems, which were a last resort against extended-spectrum β-lactamase (ESBL) producers2.

Pyrrolobenzodiazepines (PBDs) are sequence-selective DNA minor groove binding agents with antitumour6 and antibacterial7 activities. While their antitumour activities have been shown to be due to covalent DNA binding and transcription factor inhibition8, their antibacterial activities are thought to be due to a combination of DNA binding and the inhibition of DNA gyrase9. The core PBD scaffold has been described in Streptomyces species10, naturally-occurring PBDs have also been identified in Klebsiella spp11, and synthetic derivatives have demonstrated activity against Gram-negative bacteria including K. pneumoniae9. These compounds represent a potential new scaffold for antibiotic development. The antibacterial PBDs have been designed to simultaneously minimise eukaryotic toxicity whilst incorporating chemical features thought to facilitate Gram-negative entry 9,12.

Resistance to PBDs in K. pneumoniae has been observed through sequence changes in a number of genes, including those encoding Tsx and MerR-family regulator AlbA9. Tsx is an outer membrane nucleoside transporter13, and AlbA is a transcriptional regulator capable of binding antibiotics14. Both of these proteins are associated with albicidin, a DNA gyrase-targeting natural product from Xanthomonas albilineans15. The former is the mechanism by which albicidin enters the cell, and both this and the latter gene have been implicated as mechanisms of albicidin resistance in Klebsiella species13,16.

This study seeks to validate the emerging parallels between PBDs and albicidin. These are two sets of antibacterial compounds of disparate but bacterial origin, with the same molecular target, found to have the same mechanism of cell entry, and now potentially to be subject to the same resistance mechanisms. Four lead compounds were assessed for activity and resistance, to draw conclusions about whether proteins of the AlbA family are indeed responsible for sequestering antibiotics of the PBD class.

The findings in this study have significant implications for new drug discovery, given the impact of this conserved mechanism of resistance between antimicrobial classes, especially given the diversity of the chemical structures. Two additional PBDs have been synthesised in this study to understand the significance of the resistance mechanism on a range of active compounds. Additional evidence has been generated around the role of AlbA and its drug binding domain (termed AlbAS) in mediating resistance, using model systems, proteomics, and crystallography.

Results

The lead PBD compounds are active against multidrug resistant Gram-negative pathogens

We have previously described a series of PBD molecules with activity against a range of multi-drug resistant (MDR) Gram-negative and Gram-positive pathogens9; the synthesis and activity of two additional PBDs KMR-14-03 and KMR-14-14 is reported here. The PBD compounds, exemplified by KMR-14-03, KMR-14-14, KMR-14-33, and PP-A148 (Fig. 1), display promising activity against a range of Gram-negative pathogens (Table 1). Of particular note are the low MIC values against multidrug resistant Klebsiella pneumoniae and Acinetobacter baumannii strains, both priority ESKAPE pathogens, with the only exception PP-A148 against K. pneumoniae NCTC 13368. We did not see activity against the two isolates of Pseudomonas aeruginosa tested, up to a maximum concentration of 32 µg/mL. KMR-14-03 and KMR-14-33 showed good activity against all the Burkholderia species isolates tested, but no activity was seen at 32 µg/mL for KMR-14-14 with two strains: CEP509 and NCTC 10743, or with PP-A148 against C1962. Differences in the MIC values for specific compounds and in certain species or strains may reflect either influx limitations or efflux liabilities in these species, related to specific structural features.

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