In Vitro Activity Analysis of a New Polymyxin, SPR741, Tested in Combination with Antimicrobial Agents against a Challenge Set of Enterobacteriaceae, Including Molecularly Characterized Strains

Survey studies have reported that 4 to 6% of patients develop health care-associated infections (HAIs) in the United States, and Enterobacteriaceae isolates account for approximately 27% of infections (1, 2). In addition, the proportion of isolates producing extended-spectrum β-lactamases (ESBLs) have increased since 2000 and are responsible for approximately 197,400 cases and 9,100 deaths per year (3). ESBL-producing Enterobacteriaceae isolates have spread in the nosocomial and community settings, complicating the empirical treatment of infections caused by these organisms (4). The increased frequency of ESBL-producing Enterobacteriaceae isolates may increase the use of more potent antimicrobial agents (5, 6), including carbapenems, and more carbapenem use increases selection pressure for emerging and disseminating carbapenem-resistant Enterobacteriaceae (CRE) isolates (7).

Although CRE isolates are relatively uncommon in the United States, the number of U.S. facilities reporting CRE isolates has risen steadily and includes 4% of acute care hospitals and 18% of long-term acute care facilities. In addition to CRE, organisms such as Acinetobacter baumannii and other nonfermentative isolates can also express a diverse array of antimicrobial resistance mechanisms, which can compromise therapy. These often hard-to-treat infections remain one of the most pressing challenges in the infectious diseases field and drive the development of new therapeutic strategies. SPR741 is a novel polymyxin analog with no direct in vitro activity against Gram-negative bacteria, but it interacts with the outer membrane and compromises the integrity of the lipopolysaccharide. SPR741 increases cell permeability and enables antimicrobial compounds to enter (8).

We recently reported on the activity of SPR741 in combination with a series of β-lactam agents tested against a challenge set of Enterobacteriaceae (9). This study aimed to expand on the previous report and describe the in vitro activity of several more commonly used and clinically available anti-Gram-positive and tetracycline agents tested alone and in combination with SPR741 against the same challenge set of Enterobacteriaceae utilized in the previous study (9). In addition, a subset of pathogens had their genome sequenced and screened in silico for acquired macrolide-lincosamide-streptogramin (MLS) and tetracycline resistance genes for a more granular analysis.

Antimicrobial resistance challenges the empirical and guided treatment for bacterial infections and results in the concurrent use of multiple antibiotics and prolonged therapies and hospitalizations. Accordingly, the World Health Organization assembled a list of 20 major threat species (10). A report from the Centers for Disease Control and Prevention in 2013 estimated that in the United States, at least 2 million people were affected by antibiotic-resistant infections, which resulted in at least 23,000 deaths each year (11). These numbers were later confirmed to be underestimated, and revised estimates showed that more than 2.6 million antibiotic-resistant infections and nearly 44,000 deaths occurred each year when the 2013 report was released. A 2019 CDC report indicated that 2.8 million antibiotic-resistant infections occurred in the United States each year, and more than 35,000 people died as a result (3).

The current antimicrobial resistance scenario and the progress toward personalized disease treatment, including infectious diseases (12), generated an urgent need to develop new alternative approaches to treat bacterial infections. The new, expanded-spectrum polymyxin agent SPR741, in development to be used along with drugs in the current available armamentarium, represents an alternative approach for treating Gram-negative infections, including those caused by resistant organisms. Anti-Gram-positive agents, such as azithromycin, fusidic acid, and vancomycin, as tested here, are not active against Gram-negative isolates due to poor penetration through the outer membrane and/or exclusion by efflux pumps (13–15). Vancomycin is a large molecule and can only reach its target site in a Gram-negative isolate by crossing the outer membrane; however, it possesses a hydrophilic characteristic that also negates crossing. The results obtained here suggest that although SPR741 decreased the vancomycin MIC value, the cells were not permeabilized enough to reduce the MIC values around therapeutic-level concentrations, as the MIC₅₀ values remained 8 to 16 mg/liter. Azithromycin and fusidic acid also possess large molecular structures, but demonstrate a more lipophilic characteristic than vancomycin; therefore, the lower MIC values noted for azithromycin and fusidic acid when paired with SPR741 compared to vancomycin-SPR741 may be due to the lipophilic nature of the former, which may facilitate their crossing through the damaged outer membrane.

A great number of E. coli and K. pneumoniae isolates carried MLS and/or tetracycline genes, and more granular data can be seen in Table S1 in the supplemental material. The MIC results obtained by the combinations tended to correlate with the presence of these MLS and/or tetracycline genes. Regardless of the increased permeability caused by a “permeabilizer,” it is expected that increased activity by the codrug will not be observed if the bacteria possess resistance mechanisms other than decreased permeability (e.g., target site alterations/modifications). This concept was illustrated here by observing azithromycin-SPR741 MIC values against isolates carrying genes encoding ribosomal protection or posttranslational modification higher than those seen against isolates encoding esterases or phosphorylases. However, isolates carrying ribosomal protection or posttranslational modification genes were less prevalent among the isolates tested here. Similarly, isolates resistant to polymyxins were not expected to benefit from the studied combination approach. Accordingly, isolates displaying colistin MIC values of ≥4 mg/liter had MIC_50/90 results for azithromycin-SPR741, fusidic acid-SPR741, doxycycline-SPR741, and minocycline-SPR741 of 16/>32, >32/>32, 4/32, and 2/16 mg/liter, respectively, whereas the susceptible counterparts displayed MIC values of 0.5/4, 4/32, 2/32, and 0.5/2 mg/liter, respectively (Table S1).

In general, doxycycline and minocycline tested alone showed similar MIC results against these E. coli and K. pneumoniae subsets (Tables 1 and 2) and when analyzed by the presence or absence of tet genes (Table 3). Although SPR741 increased the activity of both doxycycline and minocycline against tet-negative isolates, the addition of SPR741 seemed to provide a minocycline activity greater than that observed for doxycycline. Although both molecules have similar structures, minocycline is more lipophilic than doxycycline, and this difference may favor minocycline uptake through the SPR741-damaged outer membrane (16). The presence of tet genes affected the activities of doxycycline-SPR741 and minocycline-SPR741, but the latter (4- to 8-fold) was affected to a lesser extent than doxycycline-SPR741 (16-fold). The reasons for these results are unclear, and additional investigations are needed; however, the further lipophilic nature of minocycline could also provide an advantage over doxycycline against these isolates having an outer membrane damaged by SPR741.

The results obtained here for the SPR741 combinations against E. coli isolates not carrying MLS or tet genes tended to be lower than those for K. pneumoniae, suggesting that E. coli may be more prone to permeabilization with SPR741. When overexpressed, the AcrAB-TolC system intrinsic in both species may confer resistance to a number of structurally unrelated biocides and antibiotics, including tetracyclines and glycylcyclines (17–19). In addition, decreased permeability due to alterations in porins could also play a role; however, tetracyclines seem to be less affected by alterations in porin channels (20). Moreover, azithromycin-SPR741 and fusidic acid-SPR741 MIC values tended to be higher in K. pneumoniae than E. coli, and both azithromycin and fusidic acid are recognized by the AcrAB-TolC pump. Thus, similar to tetracyclines, these MIC result differences for azithromycin-SPR741 and fusidic acid-SPR741 between K. pneumoniae and E. coli may be associated with differences in AcrAB expression levels between species. These findings emphasize a greater potential for SPR741 to permeabilize the E. coli cell envelope.

This study provides a detailed in vitro evaluation of the activity of several antimicrobial agents tested in combination with a new polymyxin, SPR741, against a challenge set of E. coli and K. pneumoniae clinical isolates. In addition, a subset of this collection was screened for MLS and tetracycline resistance genes, and these additional data allowed a more granular analysis and interpretation of results. In summary, adding SPR741 increased the in vitro activity of all tested codrugs at different levels, but the resulting activity seemed to be dependent on species, polymyxin resistance, and the biochemical properties and mechanism of resistance associated with the codrugs. However, the minocycline-SPR741 combination provided the lowest MIC₉₀ values and was somehow less affected by species, β-lactamases, or tetracycline resistance mechanisms. These results indicate that the approach evaluated here has potential for treating infections caused by E. coli and K. pneumoniae, including resistant organisms, and deserves further investigations.

We express appreciation to the following persons for significant contributions to this article: H. L. Huyhn, A. Davis, L. Deshpande, T. B. Doyle, L. Flanigan, M. Janechek, J. Oberholser, and L. N. Woosley for technical support and/or assistance with manuscript preparation.

The microbiology studies were funded by Spero Therapeutics (Cambridge, MA). JMI Laboratories also received compensation fees for services for manuscript preparation, which was also funded by Spero Therapeutics. Research reported in this publication was partially supported by BARDA, the Department of Health and Human Services Office of the Assistant Secretary for Preparedness and Response under the Cooperative Agreement no. IDSEP160030, and by an award from the Wellcome Trust as administered by CARB-X. The U.S. Army Medical Research Acquisition Activity, Fort Detrick, MD, is the awarding and administering acquisition office. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under award no. W81XWH-16-2-0019.

JMI Laboratories contracted to perform services in 2019 for Achaogen, Inc., Albany College of Pharmacy and Health Sciences, Allecra Therapeutics, Allergan, AmpliPhi Biosciences Corp., Amicrobe Advanced Biomaterials, Amplyx, Antabio, American Proficiency Institute, Arietis Corp., Arixa Pharmaceuticals, Inc., Astellas Pharma, Inc., Athelas, Basilea Pharmaceutica, Ltd., Bayer AG, Becton, Dickinson and Company, bioMérieux SA, Boston Pharmaceuticals, Bugworks Research, Inc., CEM-102 Pharmaceuticals, Cepheid, Cidara Therapeutics, Inc., CorMedix, Inc., DePuy Synthes, Destiny Pharma, Discuva, Ltd., Dr. Falk Pharma GmbH, Emery Pharma, Entasis Therapeutics, Eurofarma Laboratorios SA, U.S. Food and Drug Administration, Fox Chase Chemical Diversity Center, Inc., Gateway Pharmaceutical, LLC, GenePOC, Inc., Geom Therapeutics, Inc., GlaxoSmithKline, plc, Harvard University, Helperby, HiMedia Laboratories, F. Hoffmann-La Roche, Ltd., ICON, plc, Idorsia Pharmaceuticals, Ltd., Iterum Therapeutics plc, Laboratory Specialists, Inc., Melinta Therapeutics, Inc., Merck & Co., Inc., Microchem Laboratory, Micromyx, MicuRx Pharmaceuticals, Inc., Mutabilis Co., Nabriva Therapeutics, plc, NAEJA-RGM, Novartis AG, Oxoid, Ltd., Paratek Pharmaceuticals, Inc., Pfizer, Inc., Polyphor, Ltd., Pharmaceutical Product Development, LLC, Prokaryotics, Inc., Qpex Biopharma, Inc., Roivant Sciences, Ltd., Safeguard Biosystems, Scynexis, Inc., SeLux Diagnostics, Inc., Shionogi and Co., Ltd., SinSa Labs, Spero Therapeutics, Summit Pharmaceuticals International Corp., Synlogic, T2 Biosystems, Inc., Taisho Pharmaceutical Co., Ltd., TenNor Therapeutics, Ltd., Tetraphase Pharmaceuticals, Theravance Biopharma, University of Colorado, University of Southern California—San Diego, University of North Texas Health Science Center, VenatoRx Pharmaceuticals, Inc., Viosera Therapeutics, Vyome Therapeutics Inc., Wockhardt, Yukon Pharmaceuticals, Inc., Zai Lab, and Zavante Therapeutics, Inc. There are no speakers’ bureaus or stock options to declare. Troy Lister, Nicole Cotroneo, and Thomas R. Parr are employees of Spero Therapeutics, Inc., and may hold stock shares or stock options.

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. The contents of the article are solely the responsibility of the authors and do not necessarily represent the official views of CARB-X, the HHS Office of the Assistant Secretary for Preparedness and Response, the National Institutes of Health, or the Wellcome Trust.