scholarly journals Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Melissa D. Barnes ◽  
Marisa L. Winkler ◽  
Magdalena A. Taracila ◽  
Malcolm G. Page ◽  
Eric Desarbre ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Protein Engineering ◽  
Hydrolytic Activity ◽  
The United States ◽  
Future Design ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Hydrogen Bond Networks ◽  
Mechanistic Basis ◽  
Dynamics Simulations

ABSTRACT The emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases. IMPORTANCE Antibiotic resistance is emerging at unprecedented rates and threatens to reach crisis levels. One key mechanism of resistance is the breakdown of β-lactam antibiotics by β-lactamase enzymes. KPC-2 is a β-lactamase that inactivates carbapenems and β-lactamase inhibitors (e.g., clavulanate) and is prevalent around the world, including in the United States. Resistance to the new antibiotic ceftazidime-avibactam, which was designed to overcome KPC resistance, had already emerged within a year. Using protein engineering, we uncovered a mechanism by which resistance to this new drug emerges, which could arm scientists with the ability to forestall such resistance to future drugs.

scholarly journals Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance

2020 ◽  
pp. jbc.RA120.016461
Author(s):  
Catherine L. Tooke ◽  
Philip Hinchliffe ◽  
Robert A. Bonomo ◽  
Christopher J. Schofield ◽  
Adrian J. Mulholland ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Conformational Dynamics ◽  
Point Mutations ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Natural Variants ◽  
Activity Spectrum ◽  
Dynamics Simulations ◽  
Enzyme Intermediate

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), e.g. cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than binding (KM). High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω-, 240- and 270-loops, that border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme, and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω-loop, adopts two distinct conformations in KPC-2, either pointing ‘in’ or ‘out’ of the active site; with only the ‘in’ form compatible with deacylation. The ‘out’ conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω-loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.

scholarly journals Resurrecting Old β-Lactams: Potent Inhibitory Activity of Temocillin against Multidrug-Resistant Burkholderia Species Isolates from the United States

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Elise T. Zeiser ◽  
Scott A. Becka ◽  
Melissa D. Barnes ◽  
Magdalena A. Taracila ◽  
John J. LiPuma ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
The United States ◽  
Multidrug Resistant ◽  
Human Pathogens ◽  
Active Site Residues ◽  
Class A ◽  
Advantageous Position ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Potent Inhibitory Activity

ABSTRACT Burkholderia spp. are opportunistic human pathogens that infect persons with cystic fibrosis and the immunocompromised. Burkholderia spp. express class A and C β-lactamases, which are transcriptionally regulated by PenRA through linkage to cell wall metabolism and β-lactam exposure. The potency of temocillin, a 6-methoxy-β-lactam, was tested against a panel of multidrug-resistant (MDR) Burkholderia spp. In addition, the mechanistic basis of temocillin activity was assessed and compared to that of ticarcillin. Susceptibility testing with temocillin and ticarcillin was conducted, as was biochemical analysis of the PenA1 class A β-lactamase and AmpC1 class C β-lactamase. Molecular dynamics simulations (MDS) were performed using PenA1 with temocillin and ticarcillin. The majority (86.7%) of 150 MDR Burkholderia strains were susceptible to temocillin, while only 4% of the strains were susceptible to ticarcillin. Neither temocillin nor ticarcillin induced bla expression. Ticarcillin was hydrolyzed by PenA1 (kcat/Km = 1.7 ± 0.2 μM−1 s−1), while temocillin was slow to form a favorable complex (apparent Ki [Ki app] = ∼2 mM). Ticarcillin and temocillin were both potent inhibitors of AmpC1, with Ki app values of 4.9 ± 1.0 μM and 4.3 ± 0.4 μM, respectively. MDS of PenA revealed that ticarcillin is in an advantageous position for acylation and deacylation. Conversely, with temocillin, active-site residues K73 and S130 are rotated and the catalytic water molecule is displaced, thereby slowing acylation and allowing the 6-methoxy of temocillin to block deacylation. Temocillin is a β-lactam with potent activity against Burkholderia spp., as it does not induce bla expression and is poorly hydrolyzed by endogenous β-lactamases.

Regional outbreak of multidrug-resistant Klebsiella pneumoniae carbapenemase–producing Pseudomonas Aeruginosa

2021 ◽  
pp. 1-3
Author(s):  
Bekana K. Tadese ◽  
Anna Nutt ◽  
Ifrah Chaudhary ◽  
Charlene Offiong ◽  
Charles Darkoh
Keyword(s):  
United States ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Epidemiologic Methods ◽  
The United States ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Klebsiella Pneumoniae Carbapenemase

Abstract Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-CRPA) are rare in the United States. An outbreak of KPC-CRPA was investigated in Texas using molecular and epidemiologic methods and 17 cases of KPC-CRPA were identified. The isolates were genetically related and harbored the emerging P. aeruginosa multilocus sequence type 235, the first in the United States.

scholarly journals Monitoring Ceftazidime-Avibactam and Aztreonam Concentrations in the Treatment of a Bloodstream Infection Caused by a Multidrug-Resistant Enterobacter sp. Carrying Both Klebsiella pneumoniae Carbapenemase–4 and New Delhi Metallo-β-Lactamase–1

2019 ◽  
Vol 71 (4) ◽  
pp. 1095-1098 ◽  
Author(s):  
Mohamad Yasmin ◽  
Derrick E Fouts ◽  
Michael R Jacobs ◽  
Hanan Haydar ◽  
Steven H Marshall ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
The United States ◽  
Multidrug Resistant ◽  
Therapeutic Drug ◽  
New Delhi ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Antimicrobial Synergy ◽  
Synergy Testing ◽  
Treatment Adequacy

Abstract In an infection with an Enterobacter sp. isolate producing Klebsiella pneumoniae Carbapenemase–4 and New Delhi Metallo-β-Lactamase–1 in the United States, recognition of the molecular basis of carbapenem resistance allowed for successful treatment by combining ceftazidime-avibactam and aztreonam. Antimicrobial synergy testing and therapeutic drug monitoring assessed treatment adequacy.

scholarly journals Dissemination of the Klebsiella pneumoniae Carbapenemase in the Health Care Settings: Tracking the Trails of an Elusive Offender

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Amos Adler ◽  
Yehuda Carmeli
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Molecular Mechanisms ◽  
Antibiotic Resistance Genes ◽  
The United States ◽  
Content Type ◽  
Modes Of Transmission ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Clone Sequence ◽  
Clonal Spread

ABSTRACT Transmission of antibiotic resistance genes may be mediated by a variety of molecular mechanisms, from mobility of small genetic elements to clonal spread. Since 1997, the carbapenem-hydrolyzing enzyme Klebsiella pneumoniae carbapenemase (KPC) has spread in the United States and across the world, mainly via a single K. pneumoniae clone, sequence type 258. By tracking the trail of dissemination of the bla KPC gene inside their institution, Mathers et al. (mBio 2:e00204–11, 2011) have shown evidence of the ability of this gene to spread by several modes, including plasmid transfer and clonal spread. The ever-evolving modes of transmission of resistance genes challenge our ability to detect, track, and eventually control the spread of what has become a major threat to hospitalized patients worldwide.

Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Letícia Roloff Stallbaum ◽  
Beatriz Bohns Pruski ◽  
Suelen Cavalheiro Amaral ◽  
Stella Buchhorn de Freitas ◽  
Daniela Rodriguero Wozeak ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Multidrug Resistant ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

Transmission of novel Klebsiella pneumoniae carbapenemase-producing Escherichia coli sequence type 1193 among residents and caregivers in a community-based, residential care setting—Nevada, 2018

2020 ◽  
Vol 41 (11) ◽  
pp. 1341-1343
Author(s):  
Danica J. Gomes ◽  
Ana C. Bardossy ◽  
Lei Chen ◽  
Adrian Forero ◽  
Andrew Gorzalski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Group Home ◽  
The United States ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Sequence Type ◽  
E Coli ◽  
Residential Group ◽  
Klebsiella Pneumoniae Carbapenemase

AbstractWe describe transmission of Klebsiella pneumoniae carbapenemase-producing Escherichia coli sequence type (ST) 1193 in a group home. E. coli ST1193 is an emerging multidrug-resistant clone not previously shown to carry carbapenemases in the United States. Our investigation illustrates the potential of residential group homes to amplify rare combinations of pathogens and resistance mechanisms.

scholarly journals 603. Identification of a Carbapenemase-Producing, Extensively Drug-Resistant Klebsiella pneumoniae Isolate Carrying a blaNDM-1-Bearing, Hypervirulent Plasmid, United States 2017

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S282-S283
Author(s):  
Richard A Stanton ◽  
Gillian A McAllister ◽  
Amelia Bhatnagar ◽  
Maria Karlsson ◽  
Allison C Brown ◽  
...  
Keyword(s):  
United States ◽  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Critical Role ◽  
Illumina Miseq ◽  
The United States ◽  
Drug Resistant ◽  
Extensively Drug Resistant ◽  
Drug Classes ◽  
Carbapenemase Gene

Abstract Background The recent discovery of carbapenemase-producing hypervirulent Klebsiella pneumoniae (CP-HvKP) has signaled the convergence of multidrug resistance and pathogenicity, with the potential for increased mortality. While previous studies of CP-HvKP isolates revealed that most carried carbapenemase genes and hypervirulence elements on separate plasmids, a 2018 report from China confirmed that both could be harbored on a single, hybrid carbapenemase-hypervirulent plasmid. As part of a project sequencing isolates carrying multiple carbapenemase genes identified through CDC’s Antibiotic Resistance Laboratory Network (AR Lab Network), we discovered a blaNDM-1-bearing hypervirulent plasmid found in a KPC- and NDM-positive K. pneumoniae from the United States. Methods Antimicrobial susceptibility testing (AST) was performed by reference broth microdilution against 23 agents. Whole-genome sequencing (WGS) was performed on Illumina MiSeq and PacBio RS II platforms. Results AST results indicated the isolate was extensively drug-resistant, as it was non-susceptible to at least one agent in all but two drug classes; it was susceptible to only tigecycline and tetracycline. Analysis of WGS data showed the isolate was ST11, the same sequence type that caused a fatal outbreak of CP-HvKP in China in 2016. The genome included two plasmids. The smaller one (129kbp) carried seven antibiotic resistance (AR) genes, including the carbapenemase gene blaKPC-2. The larger plasmid (354kbp) harbored 11 AR genes, including the metallo-β-lactamase gene blaNDM-1, as well as virulence factors iucABCD/iutA, peg-344, rmpA, and rmpA2, which comprise four of the five genes previously identified as predictors of hypervirulence in K. pneumoniae. Conclusion This is the first report of a hybrid carbapenemase-hypervirulent plasmid in the United States. The presence of both blaNDM-1 and hypervirulence elements on the same plasmid suggests that the CP-Hv pathotype could spread rapidly through horizontal transfer. This discovery demonstrates the critical role of genomic characterization of emerging resistance and virulence phenotypes by the AR Lab Network as part of US containment efforts. Disclosures All authors: No reported disclosures.

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