Dataset Information

Rectal swabs are suitable for quantifying the carriage load of KPC-producing carbapenem-resistant Enterobacteriaceae.

ABSTRACT: It is more convenient and practical to collect rectal swabs than stool specimens to study carriage of colon pathogens. In this study, we examined the ability to use rectal swabs rather than stool specimens to quantify Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae (CRE). We used a quantitative real-time PCR (qPCR) assay to determine the concentration of the bla(KPC) gene relative to the concentration of 16S rRNA genes and a quantitative culture-based method to quantify CRE relative to total aerobic bacteria. Our results demonstrated that rectal swabs are suitable for quantifying the concentration of KPC-producing CRE and that qPCR showed higher correlation between rectal swabs and stool specimens than the culture-based method.

SUBMITTER: Lerner A

PROVIDER: S-EPMC3591867 | biostudies-other | 2013 Mar

REPOSITORIES: biostudies-other

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Rectal swabs are suitable for quantifying the carriage load of KPC-producing carbapenem-resistant Enterobacteriaceae.

Lerner A A Romano J J Chmelnitsky I I Navon-Venezia S S Edgar R R Carmeli Y Y

Antimicrobial agents and chemotherapy 20130107 3

It is more convenient and practical to collect rectal swabs than stool specimens to study carriage of colon pathogens. In this study, we examined the ability to use rectal swabs rather than stool specimens to quantify Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae (CRE). We used a quantitative real-time PCR (qPCR) assay to determine the concentration of the bla(KPC) gene relative to the concentration of 16S rRNA genes and a quantitative culture-based ...[more]

PMID: 23295937

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Project description: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) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-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 [Ki app] = 31 ± 3 μM) more efficiently than the K234R variant (Ki app = 270 ± 27 μM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M-1 s-1 for KPC-2 versus 247 ± 25 M-1 s-1 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 β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

Project description: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 M-1 s-1 and demonstrated a slow off-rate constant (koff) of 0.0002 s-1 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.

Dataset Information

Rectal swabs are suitable for quantifying the carriage load of KPC-producing carbapenem-resistant Enterobacteriaceae.

Publications

Rectal swabs are suitable for quantifying the carriage load of KPC-producing carbapenem-resistant Enterobacteriaceae.

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