scholarly journals 1285. Identification of KPC Omega Loop Variant R163S Conferring Ceftazidime-Avibactam Resistance

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S731-S731
Author(s):  
Alexander S Maris ◽  
Lili Tao ◽  
Paul Wada ◽  
Romney Humphries ◽  
Romney Humphries ◽  
...  
Keyword(s):  
Active Site ◽  
Tracheal Aspirate ◽  
Research Support ◽  
Carbapenem Resistant ◽  
Antigen Assay ◽  
Steady Improvement ◽  
Omega Loop ◽  
Loop Flexibility ◽  
Site Binding ◽  
Rule Out

Abstract Background We report on a 56 year-old male with prolonged COVID-19 pneumonia who initially improved with dexamethasone and intubation but quickly decompensated. Clinical and radiologic features were consistent with VAP. Tracheal aspirate cultures grew carbapenem-resistant Enterobacter cloacae; meropenem (MEM) MIC was >8 ug/ml (resistant) while ceftazidime-avibactam (CZA) MIC was 2/4 ug/ml (susceptible). Lateral flow antigen assay detected a KPC enzyme. The patient was treated with CZA with steady improvement in respiratory function over the next two weeks. He then experienced an episode of tachycardia, prompting repeat culture. At this point the patient had been extubated: sputum culture grew KPC+ E. cloacae that now showed CZA-resistance (MIC >8/4 ug/ml) and paradoxical decrease in MEM MIC (4 ug/ml); meropenem-vaborbactam (< 2/8 ug/ml) was susceptible. Methods The pre- & post-CZA therapy E. cloacae isolates underwent whole genome sequencing using the Illumina 150bp paired end protocol; sequences were quality trimmed and compared. Results A point mutation in the plasmid blaKPC3 gene was identified in the post-CZA therapy isolate, an R163S mutation in the omega loop of the enzyme. ompC and ompF porin genes were analyzed to rule-out decreased influx as a mechanism for CZA-resistance: the pre- and post-CZA isolates had identical porin sequences. Conclusion This case highlights emerging mutations within KPC carbapenemases that lead to resistance to ‘last-line’ antimicrobials like CZA. The presumptive mechanism is increased KPC active site promiscuity due to increased omega loop flexibility, allowing increased ceftazidime binding and hydrolysis, and decreased avibactam binding and beta lactamase inhibition. Paradoxically, MEM susceptibility improves after such omega loop mutations, likely due to decreased active site binding affinity, a ‘seesaw’ effect between MEM and CZA. While authors have reported MEM MICs falling into the ‘susceptible’ category after an omega loop variant, these bacteria invariably develop secondary mutations leading to MEM treatment failure. Fortunately, given our patient’s improved respiratory status, the post-CZA E. cloacae isolate was felt to reflect colonization and the patient was discharged home without antimicrobial therapy. Disclosures Romney Humphries, PhD D(ABMM), Accelerate Diagnostics (Individual(s) Involved: Self): Consultant, Shareholder; IHMA (Individual(s) Involved: Self): Consultant; Melinta (Individual(s) Involved: Self): Consultant; Momentum (Individual(s) Involved: Self): Grant/Research Support; Pattern (Individual(s) Involved: Self): Consultant; QPex (Individual(s) Involved: Self): Consultant; ThermoFisher (Individual(s) Involved: Self): Consultant; Torus (Individual(s) Involved: Self): Consultant

scholarly journals Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase

1992 ◽  
Vol 286 (1) ◽  
pp. 23-30 ◽  
Author(s):  
M F Hoylaerts ◽  
T Manes ◽  
J L Millán
Keyword(s):  
Germ Cell ◽  
Active Site ◽  
Molecular Model ◽  
Inhibition Mechanism ◽  
Side Group ◽  
Alkaline Phosphatases ◽  
Uncompetitive Inhibition ◽  
Guanidinium Group ◽  
Hydrolysis Of ◽  
Site Binding

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

scholarly journals Investigation of Carbapenemase Genes in Carbapenem Resistant Enterobacterales Isolates: First KPC Report From Dokuz Eylul University Hospital

2021 ◽  
Author(s):  
Şeyda Şilan Okalin ◽  
Ayşe Nur Sarı Kaygısız ◽  
Mahmut Cem Ergon ◽  
İbrahim Mehmet Ali Öktem
Keyword(s):  
Treatment Options ◽  
Carbapenem Resistance ◽  
Tracheal Aspirate ◽  
University Hospital ◽  
Chain Reaction ◽  
Specific Primers ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Carbapenemase Gene ◽  
Polymerase Chain

Objective: In recent years, increasing carbapenem resistance of Enterobacterales bacteria limits treatment options, considerably. The main mechanism of this resistance is the production of carbapenemase enzymes. The aim of this study is to determine carbapenemase gene types in Enterobacterales isolates from our hospitalized patients and assess the clonal associations of the isolates with KPC gene. Method: A total of 48 clinical Enterobacterales isolates resistant to at least one carbapeneme and received between January 2019 and March 2019 were included in the study. Sample types were consisted of urine, blood, tracheal aspirate, wound and sputum. Of these isolates, three were Escherichia coli while 45 were Klebsiella pneumoniae. Types of carbapenemases were investigated by polymerase chain reaction, using specific primers for VIM, IMP, NDM, KPC and OXA-48 genes. PFGE was performed to determine the clonal associations between blaKPC positive K. pnemoniae isolates. Results: According to the results, blaOXA-48 (n=2) and blaKPC (n=1) were found to be present among E. coli isolates. Regarding 45 K. pneumoniae isolates; only blaOXA-48 and only blaNDM were present in 30 and two isolates, respectively. Seven K. pneumoniae isolates were found positive for both blaOXA-48 and blaNDM. Remaining K. pneumoniae isolates (n=6) harboured only blaKPC. None of the isolates were positive for blaIMP and blaVIM. PFGE analysis showed four isolates had the same pulsotype (A), while two had different pulsotypes (B-C). Conclusion: To our knowledge, this is the first report of KPC gene isolated in Dokuz Eylul University Hospital.

A Comparison of Active Site Binding of 4-Quinolones and Novel Flavone Gyrase Inhibitors to DNA Gyrase

1995 ◽  
pp. 59-69 ◽  
Author(s):  
J. J. Hilliard ◽  
H. M. Krause ◽  
J. I. Bernstein ◽  
J. A. Fernandez ◽  
V. Nguyen ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Gyrase ◽  
Gyrase Inhibitors ◽  
Site Binding

scholarly journals Regulation of the acto·myosin subfragment 1 interaction by troponin/tropomyosin. Evidence for control of a specific isomerization between two acto·myosin subfragment 1 states

1991 ◽  
Vol 279 (3) ◽  
pp. 711-718 ◽  
Author(s):  
D F A McKillop ◽  
M A Geeves
Keyword(s):  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Thin Filaments ◽  
Closed State ◽  
Myosin Subfragment ◽  
Actomyosin Interaction ◽  
Binding Isotherms ◽  
Myosin Subfragment 1 ◽  
Site Binding

The co-operative binding of myosin subfragment 1 (S1) to reconstituted skeletal-muscle thin filaments has been examined by monitoring the fluorescence of a pyrene probe on Cys-374 of actin. The degree of co-operativity differs when phosphate, sulphate or ADP are bound to the S1 active site. Binding isotherms have been analysed according to the Geeves & Halsall [(1987) Biophys. J. 52, 215-220] model, which proposed that troponin and tropomyosin effected regulation of the actomyosin interaction by controlling an isomerization of the actomyosin complex. The data support the proposal that seven actin monomers associated with a single tropomyosin molecule act as a co-operative unit that can be in one of two states. In the ‘closed’ state myosin can bind to actin, but the subsequent isomerization is prevented. The isomerization is only allowed after the seven-actin unit is in the ‘open’ form. Ca2+ controls the proportion of actin filaments in the ‘closed’ and ‘open’ forms in the absence of myosin heads. The ratio of ‘closed’ to ‘open’ forms is approx. 50:1 in the absence of Ca2+ and 5:1 in its presence.

Active site binding modes of curcumin in HIV-1 protease and integrase

2005 ◽  
Vol 15 (14) ◽  
pp. 3364-3368 ◽  
Author(s):  
Opa Vajragupta ◽  
Preecha Boonchoong ◽  
Garrett M. Morris ◽  
Arthur J. Olson
Keyword(s):  
Active Site ◽  
Binding Modes ◽  
Hiv 1 ◽  
Site Binding

Tryptophan 500 and Arginine 707 Define Product and Substrate Active Site Binding in Soybean Lipoxygenase-1†

Biochemistry ◽  
2004 ◽  
Vol 43 (41) ◽  
pp. 13063-13071 ◽  
Author(s):  
Viola C. Ruddat ◽  
Rakesh Mogul ◽  
Ilya Chorny ◽  
Cameron Chen ◽  
Noah Perrin ◽  
...  
Keyword(s):  
Active Site ◽  
Soybean Lipoxygenase ◽  
Site Binding

Out of the active site binding pocket for carbonic anhydrase inhibitors

2015 ◽  
Vol 51 (2) ◽  
pp. 302-305 ◽  
Author(s):  
Katia D'Ambrosio ◽  
Simone Carradori ◽  
Simona M. Monti ◽  
Martina Buonanno ◽  
Daniela Secci ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Active Site ◽  
Binding Pocket ◽  
Carbonic Anhydrase Ii ◽  
Carbonic Anhydrase Inhibitors ◽  
Human Carbonic Anhydrase ◽  
Site Binding

2-Benzylsulfinylbenzoic acid binds to human carbonic anhydrase II in a mode completely different from any other class of carbonic anhydrase inhibitors investigated so far.

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