Deciphering the Efficacy of ß-Lactams in the Face of Metallo-ß-Lactamase-Derived Resistance in Enterobacterales: Supraphysiologic Zinc in the Broth Is the Culprit.

Background: In vitro-in vivo discordance in ß-lactams' activities against metallo-ß-lactamase (MBL)-producing Enterobacterales has been described. We aimed to assess whether this discordance is attributed to the supra-physiologic zinc concentration in in vitro testing media. Methods: A clinical and microbiological observational study of patients with bloodstream infections due to New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae was performed. Outcomes of patients treated empirically with non-MBL-active ß-lactam therapy (carbapenems and ceftazidime/avibactam) and MBL-active ß-lactam therapy (ceftazidime/avibactam + aztreonam) were documented. The patients' isolates were used to induce septicemia in mice, and survival upon meropenem treatment was recorded. Meropenem minimum inhibitory concentrations (MICs) were determined in standard media and in the presence of physiological zinc concentrations. Results: Twenty-nine patients receiving empiric non-MBL-active ß-lactams (median duration, 4 days) were compared with 29 receiving MBL-active ß-lactams. The 14-day mortality rates were 21% and 14%, respectively. In the murine septicemia model, meropenem treatment resulted in protection from mortality (P < .0001). Meropenem MICs in the physiologic zinc concentration broth were 1- to >16-fold lower vs MICs in zinc-unadjusted broth (≥64 mg/L). Conclusions: Our data provide foundational support to establish pharmacokinetic/pharmacodynamic relationships using MICs derived in physiologic zinc concentration, which may better predict ß-lactam therapy outcome.

Non-KPC Attributes of Newer ß-lactamase/ß-lactamase Inhibitors, Part 1: Enterobacterales and Pseudomonas aeruginosa.

Gram-negative antibiotic resistance continues to grow as a global problem due to the evolution and spread of ß-lactamases. The early ß-lactamase inhibitors (BLIs) are characterized by spectra limited to class A ß-lactamases and ineffective against carbapenemases and most extended spectrum ß-lactamases. In order to address this therapeutic need, newer BLIs were developed with the goal of treating carbapenemase producing, carbapenem resistant organisms (CRO), specifically targeting the Klebsiella pneumoniae carbapenemase (KPC). These BL/BLI combination drugs, ceftazidime/avibactam, meropenem/vaborbactam, and imipenem/relebactam, have proven to be indispensable tools in this effort. However, non-KPC mechanisms of resistance are rising in prevalence and increasingly challenging to treat. It is critical for clinicians to understand the unique spectra of these BL/BLIs with respect to non-KPC CRO. In Part 1of this two-part series, we describe the non-KPC attributes of the newer BL/BLIs with a focus on utility against Enterobacterales and Pseudomonas aeruginosa.

In vivo efficacy of WCK 6777 (ertapenem/zidebactam) against carbapenemase-producing Klebsiella pneumoniae in the neutropenic murine pneumonia model.

OBJECTIVES: Ertapenem has proven to be an effective antimicrobial; however, increasing enzyme-mediated resistance has been noted. Combination with zidebactam, a ß-lactam enhancer, is restorative. Human-simulated regimens (HSRs) of ertapenem and zidebactam alone and in combination (WCK 6777; 2 g/2 g q24h) were assessed for efficacy against carbapenemase-producing Klebsiella pneumoniae (CP-KP) in the pneumonia model. METHODS: Infected ICR mice were rendered neutropenic and exposed to various doses of ertapenem and zidebactam alone and in combination to develop the HSRs that were subsequently confirmed in additional pharmacokinetic studies. Twenty-one CP-KP (KPC or OXA-48-like producers) with WCK 6777 MICs of 1-8 mg/L were utilized. Mice were treated for 24 h with saline or HSRs of ertapenem, zidebactam and WCK 6777. Efficacy was defined as change in mean lung bacterial density relative to 0 h. RESULTS: Confirmatory pharmacokinetic analysis showed agreement between predicted human exposures (%fT>MIC) and those achieved in vivo for all three HSRs. The 0 h bacterial density across all isolates was 6.69 ±â€Š0.31 log10 cfu/lungs. At 24 h, densities increased by 2.57 ±â€Š0.50, 2.2 ±â€Š0.60 and 2.05 ±â€Š0.71 log10 cfu/lungs in the 24 h control, ertapenem HSR and zidebactam HSR groups, respectively. Overall, 18/21 of the isolates exposed to the WCK 6777 HSR displayed a killing profile that exceeded the translational benchmark for efficacy of a 1 log10 cfu reduction. Among the remaining three isolates, two displayed ∼0.5 log10 kill and stasis was observed in the third. CONCLUSIONS: Human-simulated exposures of WCK 6777 demonstrated potent in vivo activity against CP-KP, including those with WCK 6777 MICs up to 8 mg/L.

In Vitro Time-Kill Studies of Trimethoprim/Sulfamethoxazole against Stenotrophomonas maltophilia versus Escherichia coli Using Cation-Adjusted Mueller-Hinton Broth and ISO-Sensitest Broth.

Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia, but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 µg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli isolates having the same MICs (0.25/4.75 to 4/76 µg/mL) in cation-adjusted Mueller-Hinton broth (CAMHB) and ISO-Sensitest broth (ISO broth). With the exception of the resistant isolates (4/76 µg/mL), which resulted in regrowth approaching the growth of the control, TMP/SMZ displayed significantly greater killing for E. coli than for S. maltophilia at each MIC. Against E. coli, the mean changes at 24 h were -4.49, -1.73, -1.59, and +1.83 log10 CFU for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 µg/mL, respectively. The area under the concentration-time curve for the free, unbound fraction of the drug (fAUC)/MIC ratio required for stasis and 1-log10 and 2-log10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia isolate regardless of the MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.

The PX Motif of DNA Binds Specifically to Escherichia coli DNA Polymerase I.

The PX motif of DNA is a four-stranded structure in which two parallel juxtaposed double-helical domains are fused by crossovers at every point where the strands approach each other. Consequently, its twist and writhe are approximately half of those of conventional DNA. This property has been shown to relax supercoiled plasmid DNA under circumstances in which head-to-head homology exists within the plasmid; the homology can be either complete homology or every-other-half-turn homology, known as PX homology. It is clearly of interest to establish whether the cell contains proteins that interact with this unusual and possibly functional motif. We have examined Escherichia coli extracts to seek such a protein. We find by gel mobility studies that the PX motif is apparently bound by a cellular component. Fractionation of this binding activity reveals that the component is DNA polymerase I (Pol I). Although the PX motif binds to Pol I, we find that PX-DNA is not able to serve as a substrate for the extension of a shortened strand. We cannot say at this time whether the binding is a coincidence or whether it represents an activity of Pol I that is currently unknown. We have modeled the interaction of Pol I and PX-DNA using symmetry considerations and molecular dynamics.

Holey Graphene as a Weed Barrier for Molecules.

We demonstrate the use of "holey" graphene as a mask against molecular adsorption. Prepared porous graphene is transferred onto a Au{111} substrate, annealed, and then exposed to dilute solutions of 1-adamantanethiol. In the pores of the graphene lattice, we find islands of organized, self-assembled molecules. The bare Au in the pores can be regenerated by postdeposition annealing, and new molecules can be self-assembled in the exposed Au region. Graphene can serve as a robust, patternable mask against the deposition of self-assembled monolayers.