Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>

ABSTRACT Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae...

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Autores principales:	Tae Hwan Kim, Xun Tao, Bartolome Moya, Yuanyuan Jiao, Kari B. Basso, Jieqiang Zhou, Yinzhi Lang, Dhruvitkumar S. Sutaria, Alexandre P. Zavascki, Afonso L. Barth, Stephanie M. Reeve, Herbert P. Schweizer, Deanna Deveson Lucas, John D. Boyce, Robert A. Bonomo, Richard E. Lee, Beom Soo Shin, Arnold Louie, George L. Drusano, Jürgen B. Bulitta
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Enterobacter cloacae Klebsiella pneumoniae LC-MS/MS beta-lactams carbapenem resistance carbapenems Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/15920c889cbe439dba2992e6e5cf4623
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spelling	oai:doaj.org-article:15920c889cbe439dba2992e6e5cf46232021-11-15T15:56:58ZNovel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>10.1128/mBio.03189-192150-7511https://doaj.org/article/15920c889cbe439dba2992e6e5cf46232020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.03189-19https://doaj.org/toc/2150-7511ABSTRACT Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a blaKPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately (“discrete assay”) and simultaneously (“cassette assay”) by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.Tae Hwan KimXun TaoBartolome MoyaYuanyuan JiaoKari B. BassoJieqiang ZhouYinzhi LangDhruvitkumar S. SutariaAlexandre P. ZavasckiAfonso L. BarthStephanie M. ReeveHerbert P. SchweizerDeanna Deveson LucasJohn D. BoyceRobert A. BonomoRichard E. LeeBeom Soo ShinArnold LouieGeorge L. DrusanoJürgen B. BulittaAmerican Society for MicrobiologyarticleEnterobacter cloacaeKlebsiella pneumoniaeLC-MS/MSbeta-lactamscarbapenem resistancecarbapenemsMicrobiologyQR1-502ENmBio, Vol 11, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Enterobacter cloacae Klebsiella pneumoniae LC-MS/MS beta-lactams carbapenem resistance carbapenems Microbiology QR1-502
spellingShingle	Enterobacter cloacae Klebsiella pneumoniae LC-MS/MS beta-lactams carbapenem resistance carbapenems Microbiology QR1-502 Tae Hwan Kim Xun Tao Bartolome Moya Yuanyuan Jiao Kari B. Basso Jieqiang Zhou Yinzhi Lang Dhruvitkumar S. Sutaria Alexandre P. Zavascki Afonso L. Barth Stephanie M. Reeve Herbert P. Schweizer Deanna Deveson Lucas John D. Boyce Robert A. Bonomo Richard E. Lee Beom Soo Shin Arnold Louie George L. Drusano Jürgen B. Bulitta Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
description	ABSTRACT Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a blaKPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately (“discrete assay”) and simultaneously (“cassette assay”) by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.
format	article
author	Tae Hwan Kim Xun Tao Bartolome Moya Yuanyuan Jiao Kari B. Basso Jieqiang Zhou Yinzhi Lang Dhruvitkumar S. Sutaria Alexandre P. Zavascki Afonso L. Barth Stephanie M. Reeve Herbert P. Schweizer Deanna Deveson Lucas John D. Boyce Robert A. Bonomo Richard E. Lee Beom Soo Shin Arnold Louie George L. Drusano Jürgen B. Bulitta
author_facet	Tae Hwan Kim Xun Tao Bartolome Moya Yuanyuan Jiao Kari B. Basso Jieqiang Zhou Yinzhi Lang Dhruvitkumar S. Sutaria Alexandre P. Zavascki Afonso L. Barth Stephanie M. Reeve Herbert P. Schweizer Deanna Deveson Lucas John D. Boyce Robert A. Bonomo Richard E. Lee Beom Soo Shin Arnold Louie George L. Drusano Jürgen B. Bulitta
author_sort	Tae Hwan Kim
title	Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
title_short	Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
title_full	Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
title_fullStr	Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
title_full_unstemmed	Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>
title_sort	novel cassette assay to quantify the outer membrane permeability of five β-lactams simultaneously in carbapenem-resistant <italic toggle="yes">klebsiella pneumoniae</italic> and <italic toggle="yes">enterobacter cloacae</italic>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/15920c889cbe439dba2992e6e5cf4623
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Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five β-Lactams Simultaneously in Carbapenem-Resistant <italic toggle="yes">Klebsiella pneumoniae</italic> and <italic toggle="yes">Enterobacter cloacae</italic>

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