Activity Of Β-lactams Research Articles

Cysteine Hydropersulfide Inactivates β-Lactam Antibiotics with Formation of Ring-Opened Carbothioic S-Acids in Bacteria.

Hydrogen sulfide (H2S) formed during sulfur metabolism in bacteria has been implicated in the development of intrinsic resistance to antibacterial agents. Despite the conversion of H2S to hydropersulfides greatly enhancing the biochemical properties of H2S such as antioxidant activity, the effects of hydropersulfides on antibiotic resistance have remained unknown. In this work, we investigated the effects of H2S alone or together with cystine to form cysteine hydropersulfide (CysSSH) on the activities of antibacterial agents. By using the disc diffusion test, we found that CysSSH treatment effectively inactivated β-lactams of the penicillin class (penicillin G and ampicillin) and the carbapenem class (meropenem). These β-lactams were resistant to treatment with H2S alone or cystine alone. In contrast, cephalosporin class β-lactams (cefaclor and cefoperazone) and non-β-lactam antibiotics (tetracycline, kanamycin, erythromycin, and ofloxacin) were stable after CysSSH treatment. Chromatographic and mass spectrometric analyses revealed that CysSSH directly reacted with β-lactams to form β-lactam ring-opened carbothioic S-acids (BL-COSH). Furthermore, we demonstrated that certain bacteria (e.g., Escherichia coli and Staphylococcus aureus) efficiently decomposed β-lactam antibiotics to form BL-COSH, which were transported to the extracellular space. These data suggest that CysSSH-mediated β-lactam decomposition may contribute to intrinsic bacterial resistance to β-lactams. BL-COSH may become useful biomarkers for CysSSH-mediated β-lactam resistance and for investigation of potential antibacterial adjuvants that can enhance the antibacterial activity of β-lactams by reducing the hydropersulfides in bacteria.

Third-generation cephalosporin resistance in clinical isolates of Enterobacterales collected between 2016–2018 from USA and Europe: genotypic analysis of β-lactamases and comparative in vitro activity of cefepime/enmetazobactam

This study aimed to investigate third-generation cephalosporin (3GC) resistance determinants [extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and OXA-type β-lactamases] in contemporary clinical Enterobacterales isolates and to determine the in vitro activity of β-lactams and β-lactam/β-lactamase inhibitor combinations, including the investigational combination of cefepime and the novel β-lactamase inhibitor enmetazobactam. Antibacterial susceptibility of 7168 clinical Enterobacterales isolates obtained between 2016-2018 from North America and Europe was determined according to CLSI guidelines. Phenotypic resistance to the 3GC ceftazidime (MIC ≥ 16 µg/mL) and/or ceftriaxone (MIC ≥ 4 µg/mL) but retaining susceptibility to meropenem (MIC ≤ 1 µg/mL) was determined. β-Lactamase genotyping was performed on clinical isolates with ceftazidime, ceftriaxone, cefepime or meropenem MIC ≥ 1 µg/mL. Phenotypic resistance to 3GCs occurred in 17.5% of tested isolates, whereas 2.1% of isolates were resistant to the carbapenem meropenem. Within the 3GC-resistant subgroup, 60.1% (n=752) of isolates encoded an ESBL, 25.6% (n=321) encoded an AmpC-type β-lactamase and 0.9% (n=11) encoded an OXA-type β-lactamase. Susceptibility of the subgroup to piperacillin/tazobactam (57.5%) and ceftolozane/tazobactam (71.3%) was <90% based on breakpoints established by the CLSI. Projected susceptibility to cefepime/enmetazobactam was 99.6% when applying the cefepime susceptible, dose-dependent breakpoint of 8 µg/mL. Against ESBL-producing isolates (n=801) confirmed by genotyping, only susceptibility to meropenem (96.0%) and cefepime/enmetazobactam (99.9%) exceeded 90%. This study describes the antibacterial activity of important therapies against contemporary 3GC-resistant clinical Enterobacterales isolates and supports the development of cefepime/enmetazobactam as a carbapenem-sparing option for ESBL-producing pathogens.

166. Activity of a Novel β-lactamase Inhibitor QPX7728 Combined With β-lactams Against st258 klebsiella Pneumoniae and st131 escherchia Coli Isolates Producing β-lactamases

BackgroundST258 K. pneumoniae and ST131 E. coli clones are considered vectors for the global spread of multidrug resistance. We evaluated the activity of β-lactams in combination with QPX7728, a novel β-lactamase inhibitor active against all β-lactamase classes, against a collection of 210 isolates belonging to these clones collected from a worldwide surveillance study.MethodsA total of 118 ST258 K. pneumoniae and 92 ST131 E. coli (single loci variant also included) were susceptibility tested by reference broth microdilution against various β-lactams ± QPX7728 and comparator agents. All isolates were screened for β-lactamases using whole genome sequencing analysis.ResultsAll β-lactam agents had limited activity against 118 ST258 K. pneumoniae (1.7–7.6% susceptible). Among these, 104 carried carbapenemase-encoding genes: 66 KPC variants, 20 NDM and 17 OXA-48-like. One isolate carried 2 carbapenemases. The addition of QPX7728 at 4 mg/L or 8 mg/L lowered the MICs for cefepime (MIC50/90, 0.25/1 mg/L and MIC50/90, 0.12/0.5 mg/L), ceftolozane (MIC50/90, 0.5/ > 32 mg/L and MIC50/90, 0.25/16 mg/L), ertapenem (MIC50/90, 0.12/2 mg/L and MIC50/90, 0.06/0.5 mg/L), and meropenem (MIC50/90, 0.06/0.5 mg/L and MIC50/90, 0.03/0.12 mg/L; Table). QPX7728 at 4 mg/L reduced the ceftibuten (MIC50/90, 0.25/8 mg/L) or tebipenem (MIC50/90, 0.12/2 mg/L) MICs for ST258 isolates. E. coli ST131 carried mainly CTX-M variant (85 isolates), but 6 isolates harbored carbapenemases. Carbapenems were the only β-lactams displaying > 80.0% activity against ST131 E. coli, followed by piperacillin-tazobactam (79.3% susceptible). Only 5.4%and 41.3% ST131 isolates were susceptible to cefepime and ceftibuten, respectively. MIC50/MIC90 values for these agents with QPX7728 were ≤ 0.015/≤ 0.015 mg/L for cefepime and ≤ 0.015/0.06 mg/L for ceftolozane with the inhibitor at 8 mg/L and ≤ 0.015/0.03 mg/L for ceftibuten with the inhibitor at 4 mg/L.ConclusionQPX7728 lowered the MICs for all agents tested to clinically achievable levels when tested against isolates multidrug resistant belonging to important clones responsible to the dissemination of KPC, CTX variants, and metallo-β-lactamases. The development of this broad β-lactamase inhibitor should be pursued.Table 1 DisclosuresMariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Jill Lindley, Allergan (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Timothy B. Doyle, Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Olga Lomovskaya, PhD, Qpex Biopharma (Employee)

Introduction of a Thio Functional Group to Diazabicyclooctane: An Effective Modification to Potentiate the Activity of β-Lactams against Gram-Negative Bacteria Producing Class A, C, and D Serine β-Lactamases.

By the emergence and worldwide spread of multi-drug-resistant Gram-negative bacteria, there have been growing demands for efficacious drugs to cure these resistant infections. The key mechanism for resistance to β-lactam antibiotics is the production of β-lactamases, which hydrolyze and deactivate β-lactams. Diazabicyclooctane (DBO) analogs play an important role as one of the new classes of β-lactamase inhibitors (BLIs), and several compounds such as avibactam (AVI) have been approved by the FDA, along with many derivatives under clinical or preclinical development. Although these compounds have a similar amide substituent at the C2 position, we have recently reported the synthesis of novel DBO analogs which possess a thio functional group. This structural modification enhances the ability to restore the antimicrobial activities of cefixime (CMF) against pathogens producing classes A, C, and D serine β-lactamases compared with AVI and expands the structural tolerance at the six position. Furthermore, some of these analogs showed intrinsic microbial activities based on multipenicillin binding protein (PBP) inhibition. This is the unique feature which has never been observed in DBOs. One of our DBOs had a pharmacokinetic profile comparable to that of other DBOs. These results indicate that the introduction of a thio functional group into DBO is a novel and effective modification to discover a clinically useful new BLI.

Optimizing pharmacokinetics/pharmacodynamics of β-lactam/β-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria

Reduced in vitro β-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of β-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a β-lactamase inhibitor. We evaluated different β-lactam/β-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating β-lactamase inhibitor (tazobactam or avibactam) concentrations on β-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of β-lactam/β-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. A concentration-dependent reduction in β-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected β-lactam/β-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

Molecular mechanisms underlying the renoprotective effects of 1,4,7-triazacyclononane: a βeta-lactamase inhibitor.

Broad-spectrum β-lactam antibiotics such as penicillin are routinely used against both Gram-negative and Gram-positive bacteria. However, bacteria that produce β-lactamase have developed resistance against these antibiotics by cleaving the β-lactam ring and rendering the antibiotic inactive. To combat this effect, 1,4,7- Triazacyclononane (TACN), a cyclic organic compound derived from cyclononanes has been shown to preserve the activity of β-lactam antibiotics by inhibiting β-lactamase. However, its cytotoxic effects require elucidation. Given that the cytotoxic target for many therapeutics is the kidney, this study investigated the effects of TACN on human embryonic kidney cells (Hek293) cells. Hek293 cells were treated with TACN (0-500µM) for 24 h and the cytotoxicity was assessed (MTT and LDH assay). Apoptosis was luminometrically detected by measuring phosphatidylserine externalisation and caspase activity and fluorescently detecting necrosis. DNA fragmentation was visualised using fluorescent microscopy. Expression of the apoptosis-related protein were determined by western blot. The results generated indicate that TACN does not initiate necrosis as LDH was decreased. Likewise, decreased apoptosis was supported by the decreased phosphatidylserine, caspases, Bax, cleaved PARP, IAP and NF-kB. However, increased DNA fragmentation was associated with increased p53. Therefore, effects of TACN at the nucleus, produced a p53 response to initiate DNA repair and did not culminate in cell death. The findings show that TACN is not cytotoxic to Hek293 cells via the apoptotic route. Since TACN did not induce cell death, its potential as a metallo-β-lactamase inhibitor (MBLI) may be exploited to counteract the effect of MBL-producing bacteria. Restoring β-lactam activity will curb the global menace of antibiotic resistance.

4-(N-Alkyl- and -Acyl-amino)-1,2,4-triazole-3-thione Analogs as Metallo-β-Lactamase Inhibitors: Impact of 4-Linker on Potency and Spectrum of Inhibition.

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with Ki values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.

Synthesis and evaluation of biological activities of tripodal imines and β-lactams attached to the 1,3,5-triazine nucleus

A novel collection of tripodal β-lactam derivatives with 1,3,5-triazine as the central core is presented for the first time. The diastereoselective synthesis of tris-β-lactams was achieved via a [2 + 2] cycloaddition reaction between s-triazine-based tris-imines and activated aryloxyacetic acid derivatives. All the s-triazine hybrids were identified by FT-IR, 1H NMR, 13C NMR spectroscopies and elemental analysis, and found to have all-cis relative stereochemistry of the three β-lactams rings. The intermediate imines displayed moderate in vitro inhibitory activity against MCF-7 and HeLa cancer cell lines. The tris-β-lactams were tested against four kinds of bacteria including Gram-negative bacteria, E. coli and P. aeruginosa, a Gram-positive S. aureus and one capsulated Gram-negative K. pneumonia by the Kirby–Bauer disc diffusion method. Two compounds have shown moderate antibacterial activity against S. aureus, but not against the other bacteria, or fungal isolates C. albicans and A. fumigatus. The tris-β-lactams displayed good inhibitory behavior against the K562 human leukemia cell line, and antioxidant properties as radical scavengers.

In Vitro Characterization of ETX1317, a Broad-Spectrum β-Lactamase Inhibitor That Restores and Enhances β-Lactam Activity against Multi-Drug-Resistant Enterobacteriales, Including Carbapenem-Resistant Strains.

Multi-drug-resistant Enterobacteriales expressing a wide array of β-lactamases are emerging as a global health threat in both hospitals and communities. Although several intravenous drugs have recently been approved to address this need, there are no oral Gram-negative agents that are both safe and broadly effective against such pathogens. The lack of an effective oral agent is of concern for common infections which could otherwise be treated in the community but, due to antibiotic resistance, require hospitalization to allow for intravenous therapy. ETX1317 is a novel, broad spectrum, serine β-lactamase inhibitor of the diazabicyclooctane class that restores the antibacterial activity of multiple β-lactams against multiple species of multi-drug-resistant Enterobacteriales, including carbapenem-resistant strains. A combination of its oral prodrug, ETX0282, and the oral prodrug of a third-generation cephalosporin, cefpodoxime proxetil, is currently in clinical development. This report describes the biochemical and microbiological properties of ETX1317, which is more potent and demonstrates a greater breadth of inhibition than avibactam, the parenteral prototype of this class of β-lactamase inhibitors.

Diazabicyclooctane Functionalization for Inhibition of β-Lactamases from Enterobacteria

Second-generation β-lactamase inhibitors containing a diazabicyclooctane (DBO) scaffold restore the activity of β-lactams against pathogenic bacteria, including those producing class A, C, and D enzymes that are not susceptible to first-generation inhibitors containing a β-lactam ring. Here, we report optimization of a synthetic route to access triazole-containing DBOs and biological evaluation of a series of 17 compounds for inhibition of five β-lactamases representative of enzymes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; p = 4.7 × 10-21) was observed between the inhibition efficacy of purified β-lactamases and the potentiation of β-lactam antibacterial activity, indicating that DBO functionalization did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displayed reduced efficacy, likely due to the absence of hydrogen bonding with a conserved asparagine residue at position 132. This was partially compensated for by additional interactions involving certain triazole substituents.

6-Arylmethylidene Penicillin-Based Sulfone Inhibitors for Repurposing Antibiotic Efficiency in Priority Pathogens.

The ability of 6-(aryl)methylidene penicillin-based sulfones 1-7 to repurpose β-lactam antibiotics activity with bacterial species that carry carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-24/40 and OXA-48), as well as with class A (TEM-1, CTX-M-2) and class C (CMY-2, DHA-1) enzymes, is reported. The combinations imipenem/3 and imipenem/4 restored almost completely the antibiotic efficacy in OXA-23 and OXA-24/40 carbapenemase-producing A. baumannii strains (1 μg mL-1) and also provided good results for OXA-48 carbapenemase-producing K. pneumoniae strains (4 μg mL-1). Compounds 2-6 in combinations with ceftazidime and ampicillin were also efficient in restoring antibiotic efficacy in E. coli strains carrying class C (CMY-2 and DHA-1) and class A (TEM-1 and CTX-M-2) β-lactamase enzymes, respectively. Kinetic and inhibition studies with the OXA-24/40 enzyme, protein mass spectrometry analysis and docking studies allowed us to gain an insight into the inhibition mechanism and the experimentally observed differences between the ligands.

Preparation and Mechanistic Studies of 2‐Substituted Bisthiazolidines by Imine Exchange

Bisthiazolidines (BTZ) are bicyclic compounds considered penicillin analogs that inhibit the full range of Metallo-β-Lactamases (MBLs) and potentiate β-lactam activity against resistant bacteria. Herein we present a new methodology to prepare 2-substituted bisthiazolidines by aldehyde exchange. Thirteen new bisthiazolidines were prepared using this methodology, with yields ranged from 31 to 75%. The reaction is based on in situ imines formation, which are able to exchange side chains. The reaction intermediates were studied based on NMR experiments and a key imine 1b-II could be detected in the reaction mixture. Furthermore, a DFT computational analysis was performed to gain insights into the reaction mechanism, allowing us to unveil the different pathways and their activation barriers within the synthetic route. The results suggest that the most favorable route involve the formation of the thiazolidine 1b-III by i) a N-assisted N-C bond cleavage, and ii) a thiol-mediated 5 endo-trig cyclization followed by a C-N bond cleavage. In contrast with previously reported evidence, the imine metathesis was discarded as a plausible pathway. Finally, the reaction of 1b-III with aldehyde 2a leads to bicycle 4a via the iminium ion 1b-V.

Molecular Characteristics and Antimicrobial Susceptibility Profiles of Elizabethkingia Clinical Isolates in Shanghai, China.

PurposeTo investigate molecular characteristics and antimicrobial susceptibility profiles of clinical isolates of Elizabethkingia in Shanghai, China.MethodsElizabethkingia isolates were collected in a university-affiliated hospital in 2012–2015 and 2017–2018. They were re-identified to species level by 16S rRNA gene and species-specific gene sequencing. Antimicrobial susceptibility testing, screening for metallo-beta-lactamase production, identification of antimicrobial resistance genes and pulsed-field gel electrophoresis (PFGE) were performed.ResultsAmong 52 Elizabethkingia isolates, E. anophelis was the most prevalent species (67.3%), followed by E. meningoseptica (26.9%). High carriage rates of blaCME, blaBlaB and blaGOB genes were consistent with the poor in vitro activity of most β-lactams including carbapenems. Nevertheless, β-lactamase inhibitors increased susceptibility rates significantly for cefoperazone and piperacillin. Susceptibility rates for minocycline, tigecycline, rifampin and levofloxacin were 100%, 78.8%, 76.9% and 71.2%, respectively. Ser83Ile or Ser83Arg substitution in the DNA gyrase A unit was associated with resistance to fluoroquinolones. MIC50/MIC90 values of vancomycin and linezolid were 16/16 mg/L and 16/32 mg/L, respectively. Molecular typing showed twenty-one different types of PFGE and more than one indistinguishable isolates were observed in each of the eight subtypes.ConclusionTetracyclines, tigecycline, β-lactam/β-lactamase inhibitor combinations, rifampin and fluoroquinolones demonstrated high rates of in vitro activity against clinical isolates of Elizabethkingia. Both genetic diversity and clonality were observed from this health-care facility. Our report provides potential alternative treatment options for Elizabethkingia infections.

Cyclic boronates as versatile scaffolds for KPC-2 β-lactamase inhibition.

Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a serine-β-lactamase (SBL) capable of hydrolysing almost all β-lactam antibiotics. We compare KPC-2 inhibition by vaborbactam, a clinically-approved monocyclic boronate, and VNRX-5133 (taniborbactam), a bicyclic boronate in late-stage clinical development. Vaborbactam inhibition is slowly reversible, whereas taniborbactam has an off-rate indicating essentially irreversible complex formation and a 15-fold higher on-rate, although both potentiate β-lactam activity against KPC-2-expressing K. pneumoniae. High resolution X-ray crystal structures reveal closely related binding modes for both inhibitors to KPC-2, with differences apparent only in positioning of the endocyclic boronate ester oxygen. The results indicate the bicyclic boronate scaffold as both an efficient, long-lasting, KPC-2 inhibitor and capable of supporting further iterations that may improve potency against specific enzyme targets and pre-empt the emergence of inhibitor resistant KPC-2 variants.

1546. Efficacy of Daptomycin Combinations Against Daptomycin-Resistant Enterococcus faecium Differs by β-lactam

BackgroundWe have previously demonstrated that daptomycin (DAP) combinations with β-lactams offer enhanced bactericidal activity and prevent the emergence of resistance in Enterococcus faecium infections. Although the mechanisms of DAP resistance in enterococci are not fully comprehended, they are associated with alterations in cell envelope phospholipids assembly which leads to either repulsion of the drug from cell exterior or diversion from the cell septum. In this context, we sought to evaluate combinations of DAP with a panel of β-lactams including ampicillin (AMP), amoxicillin (AMX), ceftaroline (CPT), ceftriaxone (CRO) and ertapenem (ERT).Methods E. faecium R497 harboring liaSFR mutations (DAP MIC of 16 mg/L) was evaluated in a simulated endocardial vegetation (SEV) pharmacokinetic and pharmacodynamic model over 336 h at a starting inoculum of 109 log10 CFU/g. DAP 10 mg/kg/day combinations with AMP, AMX (2 g continuous infusion), CPT 600 mg q 12 h, CRO 2g q 24 h or ERT 1 g q 24 h were evaluated. The emergence of DAP resistance was determined daily over the course of treatment.ResultsDAP alone was not bactericidal and high-level DAP resistance was observed (MIC increase from 16 to 64 µg/mL). Combination of DAP+AMP offered a significant reduction in log10CFU/g amounts (Up to 7 log10 CFU/g and to detection limits) in 24h in with no emergence of DAP resistance. DAP 10+ AMX caused 6–6.5 log10 CFU/g reduction and counts were maintained around the detection limit while demonstrating no increased resistance. Dose de-escalation with AMP indicated that even DAP 4 mg/kg/d with AMP (2g) combination, reached detection limit at 168 h with no further resistance. None of the CPT, CRO or ERT regimens in combination with DAP was effective against R497 and elevated DAP MICs (>64 µg/mL) was observed during the 14-day model.ConclusionCombination of DAP+AMP offered the most encouraging results against E. faecium R497, while DAP+AMX caused enhanced reduction. The reason for this discrepancy in various β-lactam activity may be related to diverse β-lactam targeting or affinity toward PBP proteins. Further dissection of our observations is warranted to understand the optimized DAP-β-lactam combination and consequently improve patient outcomes and prevention of resistance. Disclosures All authors: No reported disclosures.

607. Scope and Predictive Genetic/Phenotypic Signatures of “Bicarbonate [NaHCO3]-Responsivity” and β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA)

BackgroundSelected MRSA strains become susceptible to β-lactams (e.g., oxacillin [OX]; cefazolin [CFZ]) in vitro when tested in a standard medium (cation-adjusted Mueller–Hinton Broth; CA-MHB) supplemented with NaHCO3 (“NaHCO3-responsivity”). In vivo activity of β-lactams was demonstrated for MRSA strains with this phenotype in a rabbit endocarditis model (Ersoy et al Antimicrob Agents Chemother 2019). The current study was designed to: (i) determine the prevalence of the NaHCO3-responsive phenotype in a large collection of clinical MRSA isolates; and (ii) identify genetic and phenotypic predictors of this phenotype. Methods. 58 recent MRSA bloodstream isolates representing contemporary clonal complex (CC) genotypes were screened for the NaHCO3-responsive phenotype by broth microdilution MICs in CA-MHB, with or without NaHCO3 supplementation (25–44 mM).Methods58 recent MRSA bloodstream isolates representing contemporary clonal complex (CC) genotypes were screened for the NaHCO3-responsive phenotype by broth microdilution MICs in CA-MHB, with or without NaHCO3 supplementation (25–44 mM).Results43/58 (74.1%) and 21/58 (36.2%) were rendered susceptible to CFZ and OX, respectively, in the presence of NaHCO3; 20 of the 21 OX-susceptible strains were also susceptible to CFZ in the presence of NaHCO3. High baseline β-lactam MICs (i.e., MICs in CA-MHB alone ≥64 µg/mL) was not predictive of NaHCO3 responsivity. The CC8 genotype was correlated with NaHCO3 responsivity for OX, but not CFZ (P < 0.05).ConclusionThe NaHCO3-responsive phenotype is relatively common for both OX and especially CFZ among clinical MRSA isolates. Identification of specific genetic factors linked to this phenotype remains ongoing. Confirmation in relevant animal models that this phenotype is predictive of β-lactam efficacy in vivo could provide a solid foundation for a paradigm shift in antimicrobial susceptibility testing of MRSA.Disclosures All authors: No reported disclosures.

715. Bulgecin A lowers Imipenem MICs in Clinical MDR Acinetobacter baumannii (ACB) Strains

BackgroundGenetic screening of ACB has revealed genes that confer increased susceptibility to β-lactams when these genes are disrupted suggesting novel drug targets. One such target is lytic transglycosylase, LT. Bulgecin A (BlgA) is a natural product of Paraburkholderia acidophila and an LT inhibitor that potentiates the activity of β-lactams.MethodsBroth microdilution MICs were performed using carbapenem-resistant (CR)-colisitin susceptible (ColS) and CR-ColR clinical ACB strains, with a variety of resistance mechanisms, previously studied via whole-genome sequencing. A fixed concentration of pure BlgA at 100 μg/mL was combined with varying concentrations of imipenem. Sequences of the putative LTs in ACB strains were analyzed to look for amino-acid substitutions and correlated with the MIC lowering effect of BlgA. Homology models of the LTs of AB0057 ACB strain were generated.ResultsACB MltE most resembles soluble LTs of other species with 22.39% sequence identity and 92% query coverage to Slt70 of E. coli. MIC results and amino-acid sequence variations in MltE LT of ACB are shown. There were no clear amino-acid substitution patterns to account for differences in BlgA effect. The mutations are distal to the BlgA binding site at residue E506.ConclusionThese are the first MICs reported for BlgA in combination with a carbapenem demonstrating reduction in the MIC by four-fold for several MDR ACB clinical strains. Mutations in MltE Lt do not account for variations in the MICs observed. Changes in the MICs may be related to other factors such as drug penetration and alterations in PBP expression. Additional studies are underway to examine these factors. Disclosures All authors: No reported disclosures.

Impact of relebactam-mediated inhibition of Mycobacterium abscessus BlaMab β-lactamase on the in vitro and intracellular efficacy of imipenem.

Imipenem is one of the recommended β-lactams for the treatment of Mycobacterium abscessus pulmonary infections in spite of the production of BlaMab β-lactamase. Avibactam, a second-generation β-lactamase inhibitor, was previously shown to inactivate BlaMab, but its partner drug, ceftazidime, is devoid of any antibacterial activity against M. abscessus. Here, we investigate whether relebactam, a novel second-generation inhibitor developed in combination with imipenem, improves the activity of this carbapenem against M. abscessus. The impact of BlaMab inhibition by relebactam was evaluated by determining MICs, time-kill curves and M. abscessus intracellular proliferation in human macrophages. Kinetic parameters for the inhibition of BlaMab by relebactam were determined by spectrophotometry using nitrocefin as the substrate. The data were compared with those obtained with avibactam. Combination of relebactam (4 mg/L) with β-lactams led to >128- and 2-fold decreases in the MICs of amoxicillin (from >4096 to 32 mg/L) and imipenem (from 8 to 4 mg/L). In vitro, M. abscessus was not killed by the imipenem/relebactam combination. In contrast, relebactam increased the intracellular activity of imipenem, leading to 88% killing. Relebactam and avibactam similarly potentiated the antibacterial activities of β-lactams although BlaMab was inactivated 150-fold less effectively by relebactam than by avibactam. Inhibition of BlaMab by relebactam improves the efficacy of imipenem against M. abscessus in macrophages, indicating that the imipenem/relebactam combination should be clinically considered for the treatment of infections due to M. abscessus.

Semimechanistic Pharmacodynamic Modeling of Aztreonam-Avibactam Combination to Understand Its Antimicrobial Activity Against Multidrug-Resistant Gram-Negative Bacteria.

Aztreonam‐avibactam (ATM‐AVI) is a promising combination to treat serious infections caused by multidrug‐resistant (MDR) pathogens. Three distinct mechanisms of action have been previously characterized for AVI: inhibition of ATM degradation by β‐lactamases, proper bactericidal effect, and enhancement of ATM bactericidal activity. The aim of this study was to quantify the individual contribution of each of the three AVI effects. In vitro static time‐kill studies were performed on four MDR Enterobacteriaceae with different β‐lactamase profiles. β‐Lactamase activity was characterized by measuring ATM concentrations over 27 hours. Data were analyzed by a semimechanistic pharmacodynamics modeling approach. Surprisingly, even though AVI prevented ATM degradation, the combined bactericidal activity was mostly explained by the enhancement of ATM effect within clinical range of ATM (5–125 mg/L) and AVI concentrations (0.9–22.5 mg/L). Therefore, when selecting a β‐lactamase inhibitor for combination with a β‐lactam, its capability to enhance the β‐lactam activity should be considered in addition to the spectrum of β‐lactamases inhibited.

Bedaquiline Eliminates Bactericidal Activity of β-Lactams against Mycobacterium abscessus.

The β-lactams imipenem and cefoxitin are used for the treatment of Mycobacterium abscessus lung infections. Here, we show that these cell wall synthesis inhibitors trigger a lethal bacterial ATP burst by increasing oxidative phosphorylation. Cotreatment of M. abscessus with the antimycobacterial ATP synthase inhibitor bedaquiline suppresses this ATP burst and eliminates the bactericidal activity of β-lactams. Thus, the addition of bedaquiline to β-lactam-containing regimes may negatively affect treatment outcome.