Efficacy of SPR720 in murine models of non-tuberculous mycobacterial pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is increasing worldwide, with Mycobacterium avium complex (MAC) and Mycobacterium abscessus as the predominant pathogens. Current treatments are poorly tolerated and modestly effective, highlighting the need for new treatments. SPR719, the active moiety of the benzimidazole prodrug SPR720, inhibits the ATPase subunits of DNA gyrase B, a target not exploited by current antibiotics, and therefore, no cross-resistance is expected with standard-of-care (SOC) agents. OBJECTIVES: To evaluate the in vitro activity of SPR719 against MAC and M. abscessus clinical isolates, including those resistant to SOC agents, and in vivo efficacy of SPR720 in murine non-tuberculous mycobacteria (NTM) pulmonary infection models. METHODS: NTM isolates were tested for susceptibility to SPR719. Chronic C3HeB/FeJ and severe combined immunodeficient murine models of pulmonary infection were used to assess efficacy of SPR720 against MAC and M. abscessus, respectively. RESULTS: SPR719 was active against MAC (MIC90, 2 mg/L) and M. abscessus (MIC90, 4 mg/L) clinical isolates. Efficacy of SPR720 was demonstrated against MAC pulmonary infection, both as a monotherapy and in combination with SOC agents. SPR720 monotherapy exhibited dose-dependent reduction in bacterial burden, with the largest reduction observed when combined with clarithromycin and ethambutol. Efficacy of SPR720 was also demonstrated against M. abscessus pulmonary infection where monotherapy exhibited a dose-dependent reduction in bacterial burden with further reductions detected when combined with SOC agents. CONCLUSIONS: In vitro activity of SPR720 against common NTM pathogens and efficacy in murine infections warrant the continued clinical evaluation of SPR720 as a new oral option for the treatment of NTM-PD.

Contemporary Evaluation of Tebipenem In Vitro Activity against Enterobacterales Clinical Isolates Causing Urinary Tract Infections in US Medical Centers (2019-2020).

Tebipenem pivoxil is an oral broad-spectrum carbapenem. This study evaluated the activity of tebipenem and comparators against UTI Enterobacterales from US hospitals (2019-2020). 3,576 Enterobacterales causing UTI in 52 centers in 9 US Census Divisions were included. Susceptibility testing followed the CLSI broth microdilution method. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis with an MIC of ≥2 µg/mL for ceftazidime, ceftriaxone, and/or aztreonam were designated ESBL. Isolates were also grouped based on MDR phenotype. Tebipenem, meropenem, and ertapenem had MIC90 against Enterobacterales of 0.06 µg/mL, 0.06 µg/mL and 0.03 µg/mL, respectively. Low susceptibility results for aztreonam (87.1% susceptible), cefazidime (88.1%), ceftriaxone (84.8%), and other agents were observed. Tebipenem and ertapenem were equally potent (MIC90, 0.015 to 0.03 µg/mL) against E. coli and K. pneumoniae, whereas ertapenem showed an MIC 8-fold lower than tebipenem against P. mirabilis. Oral agents, such as amoxicillin-clavulanate, levofloxacin, and trimethoprim-sulfamethoxazole, showed elevated nonsusceptibility rates in the Middle Atlantic region (26, 45, 47, and 41%, respectively). ESBL prevalence varied from 7% to 16%, except in the Middle Atlantic region (42%). The carbapenems were active against ESBL and MDR isolates (93.7 to 96.8% susceptible). Elevated rates of ESBL in UTI pathogens in US hospitals were noted as well as a uniform in vitro potency (MIC90) of tebipenem and the intravenous carbapenems, regardless of phenotype. IMPORTANCE The occurrence of urinary-tract Enterobacterales pathogens producing ESBL enzymes in community and nosocomial settings continues to increase, as does the coresistance to fluoroquinolones, trimethoprim-sulfamethoxazole and nitrofurantoin often exhibited by these pathogens. This scenario complicates the clinical empirical and guided management of UTI by precluding the use of oral and many intravenous options. Oral options appear compromised even among some ESBL-negative isolates, against which the use of parenteral agents may be required. In addition, the interregional variability of susceptibility results of US UTI pathogens provides a less predictable susceptibility pattern to inform empirical treatment decisions. This study evaluated the in vitro activity of tebipenem against contemporary uropathogens, including those resistant to currently available oral options.

In Vitro Activity of Tebipenem against Various Resistant Subsets of Escherichia coli Causing Urinary Tract Infections in the United States (2018 to 2020).

This study investigated the activity of an oral carbapenem, tebipenem, against various molecularly characterized subsets of Escherichia coli. A total of 15.0% of E. coli isolates (360/2,035 isolates) met the MIC criteria for screening for ß-lactamases. Most of those isolates (74.7% [269/360 isolates]) carried blaCTX-M. The CTX-M distribution varied (50% to 86%) among Census Regions, as did that of plasmid AmpC genes (up to 41% among E. coli isolates from the New England Region). Tebipenem and intravenous carbapenems showed uniform activity against various E. coli subsets.

Evaluation of Tebipenem Hydrolysis by ß-Lactamases Prevalent in Complicated Urinary Tract Infections.

Tebipenem pivoxil is the first orally available carbapenem antibiotic and has been approved in Japan for treating ear, nose, and throat and respiratory infections in pediatric patients. Its active moiety, tebipenem, has shown potent antimicrobial activity in vitro against clinical isolates of Enterobacterales species from patients with urinary tract infections (UTIs), including those producing extended-spectrum ß-lactamases (ESBLs) and/or AmpC ß-lactamase. In the present study, tebipenem was tested for stability to hydrolysis by a set of clinically relevant ß-lactamases, including TEM-1, AmpC, CTX-M, OXA-48, KPC, and NDM-1 enzymes. In addition, hydrolysis rates of other carbapenems, including imipenem, meropenem, and ertapenem, were determined for comparison. It was found that, similar to other carbapenems, tebipenem was resistant to hydrolysis by TEM-1, CTX-M, and AmpC ß-lactamases but was susceptible to hydrolysis by KPC, OXA-48, and NDM-1 enzymes with catalytic efficiency values (kcat/Km) ranging from 0.1 to 2 × 106 M-1s-1. This supports the reported results of antimicrobial activity of tebipenem against ESBL- and AmpC-producing but not carbapenemase-producing Enterobacterales isolates. Considering that CTX-M and AmpC ß-lactamases represent the primary determinants of multidrug-resistant complicated UTIs (cUTIs), the stability of tebipenem to hydrolysis by these enzymes supports the utility of its prodrug tebipenem, tebipenem pivoxil hydrobromide (TBP-PI-HBr), as an oral therapy for adult cUTIs.

In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus.

The aminobenzimidazole SPR719 targets DNA gyrase in Mycobacterium tuberculosis. The molecule acts as inhibitor of the enzyme's ATPase located on the Gyrase B subunit of the tetrameric Gyrase A2B2 protein. SPR719 is also active against non-tuberculous mycobacteria (NTM) and recently entered clinical development for lung disease caused by these bacteria. Resistance against SPR719 in NTM has not been characterized. Here, we determined spontaneous in vitro resistance frequencies in single step resistance development studies, MICs of resistant strains, and resistance associated DNA sequence polymorphisms in two major NTM pathogens Mycobacterium avium and Mycobacterium abscessus. A low-frequency resistance (10-8/CFU) was associated with missense mutations in the ATPase domain of the Gyrase B subunit in both bacteria, consistent with inhibition of DNA gyrase as the mechanism of action of SPR719 against NTM. For M. abscessus, but not for M. avium, a second, high-frequency (10-6/CFU) resistance mechanism was observed. High-frequency SPR719 resistance was associated with frameshift mutations in the transcriptional repressor MAB_4384 previously shown to regulate expression of the drug efflux pump system MmpS5/MmpL5. Our results confirm DNA gyrase as target of SPR719 in NTM and reveal differential resistance development in the two NTM species, with M. abscessus displaying high-frequency indirect resistance possibly involving drug efflux. IMPORTANCE Clinical emergence of resistance to new antibiotics affects their utility. Characterization of in vitro resistance is a first step in the profiling of resistance properties of novel drug candidates. Here, we characterized in vitro resistance against SPR719, a drug candidate for the treatment of lung disease caused by non-tuberculous mycobacteria (NTM). The identified resistance associated mutations and the observed differential resistance behavior of the two characterized NTM species provide a basis for follow-up studies of resistance in vivo to further inform clinical development of SPR719.

Resistance among urinary tract pathogens collected in Europe during 2018.

OBJECTIVES: Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis are urinary tract infection (UTI) pathogens and extended spectrum ß-lactamase (ESBL)-producing pathogens exhibit co-resistance to oral fluoroquinolones (FQ) and trimethoprim-sulphamethoxazole (TMP-SMX). This study assessed the prevalence of ESBL phenotypes and co-resistance to FQ and TMP-SMX. METHODS: In total, 766 E. coli, 260 K. pneumoniae and 104 P. mirabilis from UTIs in 18 countries were evaluated for susceptibility in the SENTRY surveillance programme, and results interpreted using EUCAST criteria. RESULTS: E. coli, K. pneumoniae and P. mirabilis accounted for 57.1%, 11.3% and 7.8%, respectively, of the isolates. Among E. coli, resistance to levofloxacin and TMP-SMX ranged from 21.8% to 32.7% for all isolates increasing to 66.5-67.0% among those with a ESBL phenotype (17.9% of all UTI E. coli from Europe were ESBL phenotypes). In contrast, all E. coli were susceptible to meropenem. For K. pneumoniae, resistance rates for levofloxacin and TMP-SMX were 32.2-40.0% increasing to 69.1-78.6% for ESBL phenotypes. Meropenem was the most active agent, with 7.7% resistance. Among P. mirabilis resistance to levofloxacin and TMP-SMX was 26-38.5% and increased to 100% for ESBL phenotypes. No meropenem-resistant P. mirabilis were reported. CONCLUSIONS: High co-resistance rates were observed for oral antibiotics among ESBL phenotypes raising concerns regarding empiric use of FQ and TMP-SMX for treating resistant UTIs outside of the hospital. In contrast, intravenous carbapenems retain activity against resistant UTI pathogens. New oral options with the spectrum of the carbapenems would address an unmet need for managing resistant UTIs.

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.

The activities of azithromycin, fusidic acid, vancomycin, doxycycline, and minocycline were evaluated alone and in combination with SPR741. A total of 202 Escherichia coli and 221 Klebsiella pneumoniae isolates were selected, and they included a genome-sequenced subset (n = 267), which was screened in silico for ß-lactamase, macrolide-lincosamide-streptogramin (MLS), and tetracycline (tet) genes. Azithromycin (>16 mg/liter), fusidic acid (>64 mg/liter), vancomycin (>16 mg/liter), and SPR741 (>8 mg/liter) showed off-scale MICs when each was tested alone against all isolates. MIC50/90 results of 0.5/8 mg/liter, 4/>32 mg/liter, 16/>16 mg/liter, 2/32 mg/liter, and 0.25/4 mg/liter were obtained for azithromycin-SPR741, fusidic acid-SPR741, vancomycin-SPR741, doxycycline-SPR741 and minocycline-SPR741, respectively, against all isolates. Overall, azithromycin-SPR741 (MIC90, 2 to 4 mg/liter) and minocycline-SPR741 (MIC90, 0.5 to 2 mg/liter) showed the lowest MIC90 values against different subsets of E. coli isolates, except for azithromycin-SPR741 (MIC90, 16 mg/liter) against the AmpC and metallo-ß-lactamase subsets. In general, minocycline-SPR741 (MIC90, 2 to 8 mg/liter) had the lowest MIC90 against K. pneumoniae isolates producing different groups of ß-lactamases. The azithromycin-SPR741 MIC (MIC50/90, 2/32 mg/liter) was affected by MLS genes (MIC50/90 of 0.25/2 mg/liter against isolates without MLS genes), whereas doxycycline-SPR741 (MIC50/90, 0.5/2 versus 8/32 mg/liter) and minocycline-SPR741 (MIC50/90, 0.25/1 versus 1/8 mg/liter) MIC results were affected when tested against isolates carrying tet genes in general. However, minocycline-SPR741 inhibited 88.2 to 92.9% of tet-positive isolates regardless of the tet gene. The azithromycin-SPR741 MIC results (MIC50/90, 1/16 mg/liter) against isolates with enzymatic MLS mechanisms were lower than against those with ribosomal protection (MIC50/90, 16/>32 mg/liter). SPR741 increased the in vitro activity of tested codrugs at different levels and seemed to be dependent on the species and resistance mechanisms of the respective codrug.

In Vitro and In Vivo Characterization of Tebipenem, an Oral Carbapenem.

The continued evolution of bacterial resistance to the ß-lactam class of antibiotics has necessitated countermeasures to ensure continued effectiveness in the treatment of infections caused by bacterial pathogens. One relatively successful approach has been the development of new ß-lactam analogs with advantages over prior compounds in this class. The carbapenems are an example of such ß-lactam analogs possessing improved stability against ß-lactamase enzymes and, therefore, a wider spectrum of activity. However, all carbapenems currently marketed for adult patients are intravenous agents, and there is an unmet need for an oral agent to treat patients that otherwise do not require hospitalization. Tebipenem pivoxil hydrobromide (tebipenem-PI-HBr or SPR994) is an orally available prodrug of tebipenem, a carbapenem with activity versus multidrug-resistant (MDR) Gram-negative pathogens, including quinolone-resistant and extended-spectrum-ß-lactamase-producing Enterobacterales Tebipenem-PI-HBr is currently in development for the treatment of complicated urinary tract infections (cUTI). Microbiological data are presented here that demonstrate equivalency of tebipenem with intravenous carbapenems such as meropenem and support its use in infections in which the potency and spectrum of a carbapenem are desired. The results from standard in vitro microbiology assays as well as efficacy in several in vivo mouse infection models suggest that tebipenem-PI-HBr could be a valuable oral agent available to physicians for the treatment of infections, particularly those caused by antibiotic-resistant Gram-negative pathogens.

Potentiation of Antibiotics against Gram-Negative Bacteria by Polymyxin B Analogue SPR741 from Unique Perturbation of the Outer Membrane.

Therapeutics targeting Gram-negative bacteria have the challenge of overcoming a formidable outer membrane (OM) barrier. Here, we characterize the action of SPR741, a novel polymyxin B (PMB) analogue shown to potentiate several large-scaffold antibiotics in Gram-negative pathogens. Probing the surface topology of Escherichia coli using atomic force microscopy revealed substantial OM disorder at concentrations of SPR741 that lead to antibiotic potentiation. Conversely, very little cytoplasmic membrane depolarization was observed at these same concentrations, indicating that SPR741 acts predominately on the OM. Truncating the lipopolysaccharide (LPS) core with genetic perturbations uniquely sensitized E. coli to SPR741, suggesting that LPS core residues keep SPR741 at the OM, where it can potentiate a codrug, rather than permit its entry to the cytoplasmic membrane. Further, a promoter activity assay revealed that SPR741 challenge induced the expression of RcsAB, a stress sensor for OM perturbation. Together, these results indicate that SPR741 interacts predominately with the OM, in contrast to the dual action of PMB and colistin at both the outer and cytoplasmic membranes.

Evaluation of Antimicrobial Effects of a New Polymyxin Molecule (SPR741) When Tested in Combination with a Series of ß-Lactam Agents Against a Challenge Set of Gram-Negative Pathogens.

The antimicrobial activities of several ß-lactam agents were tested by broth microdilution alone and in combination with a new polymyxin analog, SPR741 (at a fixed concentration of 8 mg/L), against a challenge set of clinical isolates (202 Escherichia coli and 221 Klebsiella pneumoniae isolates). Using Clinical and Laboratory Standards Institute (CLSI) or European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility criteria for each partner antibiotic, mecillinam-SPR741, temocillin-SPR741, and piperacillin-tazobactam-SPR741 combinations had susceptibility rates higher (85.6-100.0%) than the respective agents tested alone (47.5-88.7%) against extended-spectrum ß-lactamase (ESBL)-producing E. coli and K. pneumoniae. Temocillin-SPR741 (97.8% susceptible) had MIC50 (minimum inhibitory concentration) and MIC90 results of 0.5 and 2 mg/L, respectively, against K. pneumoniae carbapenemase (KPC)-producing E. coli, 8- to 16-fold lower than temocillin alone (MIC50/90, 8/16 mg/L; 65.2% susceptible). The mecillinam MIC50/MIC90 results dropped to 1/4 mg/L (from 128/>256 mg/L when tested alone) against metallo-ß-lactamase (MBL)-producing E. coli. These MICs for mecillinam-SPR741 resulted in a susceptibility rate of 96.9% versus 9.4% for mecillinam. In general, a decrease in MICs for ß-lactams (MIC90, >32 mg/L) in the presence of SPR741 was not observed against KPC-, MBL- or OXA-48-like-producing K. pneumoniae. These study results indicate that some agents had a significant increase in in vitro activity in the presence of SPR741 and could become potential strategic options for treating serious infections caused by multidrug-resistant Enterobacteriaceae.

The burden of antimicrobial resistance among urinary tract isolates of Escherichia coli in the United States in 2017.

Urinary tract infections (UTIs) caused by Escherichia coli have been historically managed with oral antibiotics including the cephalosporins, fluoroquinolones and trimethoprim-sulfamethoxazole. The use of these agents is being compromised by the increase in extended spectrum ß-lactamase (ESBL)-producing organisms, mostly caused by the emergence and clonal expansion of E. coli multilocus sequence typing (ST) 131. In addition, ESBL isolates show co-resistance to many of oral agents. Management of UTIs caused by ESBL and fluoroquinolone-resistant organisms is becoming increasingly challenging to treat outside of the hospital setting with clinicians having to resort to intravenous agents. The aim of this study was to assess the prevalence of ESBL phenotypes and genotypes among UTI isolates of E. coli collected in the US during 2017 as well as the impact of co-resistance to oral agents such as the fluoroquinolones and trimethoprim-sulfamethoxazole. The national prevalence of ESBL phenotypes of E. coli was 15.7% and was geographically distributed across all nine Census regions. Levofloxacin and trimethoprim-sulfamethoxazole-resistance rates were ≥ 24% among all isolates and this co-resistance phenotype was considerably higher among isolates showing an ESBL phenotype (≥ 59.2%) and carrying blaCTX-M-15 (≥ 69.5%). The agents with the highest potency against UTI isolates of E. coli, including ESBL isolates showing cross-resistance across oral agents, were the intravenous carbapenems. The results of this study indicate that new oral options with the spectrum and potency similar to the intravenous carbapenems would address a significant unmet need for the treatment of UTIs in an era of emergence and clonal expansion of ESBL isolates resistant to several classes of antimicrobial agents, including oral options.

Pharmacodynamics of Tebipenem: New Options for Oral Treatment of Multidrug-Resistant Gram-Negative Infections.

Tebipenem pivoxil HBr (TBPM-PI-HBr) is a novel orally bioavailable carbapenem. The active moiety is tebipenem. Tebipenem pivoxil is licensed for use in Japan in children with ear, nose, and throat infections and respiratory infections. The HBr salt was designed to improve drug substance and drug product properties, including stability. TBPM-PI-HBr is now being developed as an agent for the treatment of complicated urinary tract infections (cUTI) in adults. The pharmacokinetics-pharmacodynamics of tebipenem were studied in a well-characterized neutropenic murine thigh infection model. Plasma drug concentrations were measured using liquid chromatography-tandem mass spectrometry. Dose fractionation experiments were performed after establishing dose-response relationships. The magnitude of drug exposure required for stasis was established using 11 strains of Enterobacteriaceae (Escherichia coli, n = 6; Klebsiella pneumoniae, n = 5) with a variety of resistance mechanisms. The relationship between drug exposure and the emergence of resistance was established in a hollow-fiber infection model (HFIM). Tebipenem exhibited time-dependent pharmacodynamics that were best described by the free drug area under the concentration-time curve (fAUC0-24)/MIC corrected for the length of the dosing interval (fAUC0-24/MIC · 1/tau). The pharmacodynamics of tebipenem versus E. coli and K. pneumoniae were comparable, as was the response of strains possessing extended-spectrum ß-lactamases versus the wild type. The median fAUC0-24/MIC · 1/tau value for the achievement of stasis in the 11 strains was 23. Progressively more fractionated regimens in the HFIM resulted in the suppression of resistance. An fAUC0-24/MIC · 1/tau value of 34.58 to 51.87 resulted in logarithmic killing and the suppression of resistance. These data and analyses will be used to define the regimen for a phase III study of adult patients with cUTI.

Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms.

The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum ß-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.

Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus.

The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.