Relebactam is a potent inhibitor of the kpc-2 -lactamase and restores imipenem susceptibility in kpc-producing enterobacteriaceae

The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex -lactamase backgrounds. Relebactam is a -lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae. MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae (K. pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Citrobacter koseri, and Escherichia coli) were highly susceptible to imipenem-relebactam (MICs 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant (k2/K) value of 24,750 M1 s1 and demonstrated a slow off-rate constant (koff) of 0.0002 s1. Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known

Nacubactam enhances meropenem activity against carbapenem-resistant klebsiella pneumoniae producing KPC

Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) -lactamase inhibitor that inactivates class A and C -lactamases and exhibits intrinsic antibiotic and -lactam “enhancer” activity against Enterobacteriaceae. In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Kiapp] 31 3 M) more efficiently than the K234R variant (Kiapp 270 27 M) and displayed a faster acylation rate (k2/K), which was 5,815 582 M1 s1 for KPC-2 versus 247 25 M1 s1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam’s aminoethoxy tail formed unproductive interactions with the K234R variant’s active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae. Moreover, our data suggest that -lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam

The BioWipe: a non-invasive method to detect intestinal carriage of multi-drug resistant GRAM-negative bacteria

Colonization precedes infection and facilitates spread of several clinically important multidrug resistant organisms (MDRO). Reliable detection of carriage is important to improve our understanding of risk factors and spread of MDRO. Bacterial culture of stool samples obtained from peri-rectal swabs or whole stool is often used for this purpose. The previously described BioWipe method is a non-invasive stool collection method that resembles the use of toilet paper, and can be self-administered. The BioWipe consists of a 100×160 mm square of soft, absorbent synthetic fiber material attached to a plastic backing layer (Fisher Scientific, USA). It is used prior to using toilet paper after a bowel movement. The wipe with collected stool sample is placed onto the surface of an absorbent pad (3M™ Petroleum Sorbent Pads, Fisher Scientific, USA) containing modified Cary Blair transport media. The two parts are then folded together and placed inside a plastic bag. Prior to use, both components are treated with ultraviolet light irradiation in a biological safety cabinet for 30 minutes. After sample collection, the BioWipe is eluted with 20 mL mix of Phosphate Buffer Saline solution (PBS, pH=7.2) and 0.1% Tween 80 (vol/vol) directly in its original bag in a biosafety cabinet, until the stool sample is completely eluted. The resulting suspended stool sample is used for further processing