Activity of ceftolozane/tazobactam and imipenem/relebactam against clinical isolates of Enterobacterales and Pseudomonas aeruginosa collected in Greece and Italy-SMART 2017-2021.

PURPOSE: The current study evaluated the in vitro activities of ceftolozane/tazobactam (C/T), imipenem/relebactam (IMI/REL), and comparators against recent (2017-2021) clinical isolates of gram-negative bacilli from two countries in southern Europe. METHODS: Nine clinical laboratories (two in Greece; seven in Italy) each collected up to 250 consecutive gram-negative isolates per year from lower respiratory tract, intraabdominal, urinary tract, and bloodstream infection samples. MICs were determined by the CLSI broth microdilution method and interpreted using 2022 EUCAST breakpoints. ß-lactamase genes were identified in select ß-lactam-nonsusceptible isolate subsets. RESULTS: C/T inhibited the growth of 85-87% of Enterobacterales and 94-96% of ESBL-positive non-CRE NME (non-Morganellaceae Enterobacterales) isolates from both countries. IMI/REL inhibited 95-98% of NME, 100% of ESBL-positive non-CRE NME, and 98-99% of KPC-positive NME isolates from both countries. Country-specific differences in percent susceptible values for C/T, IMI/REL, meropenem, piperacillin/tazobactam, levofloxacin, and amikacin were more pronounced for Pseudomonas aeruginosa than Enterobacterales. C/T and IMI/REL both inhibited 84% of P. aeruginosa isolates from Greece and 91-92% of isolates from Italy. MBL rates were estimated as 4% of Enterobacterales and 10% of P. aeruginosa isolates from Greece compared to 1% of Enterobacterales and 3% of P. aeruginosa isolates from Italy. KPC rates among Enterobacterales isolates were similar in both countries (7-8%). OXA-48-like enzymes were only identified in Enterobacterales isolates from Italy (1%) while GES carbapenemase genes were only identified in P. aeruginosa isolates from Italy (2%). CONCLUSION: We conclude that C/T and IMI/REL may provide viable treatment options for many patients from Greece and Italy.

Activity of ceftolozane/tazobactam and imipenem/relebactam against clinical isolates of Enterobacterales and Pseudomonas aeruginosa collected in central and northern Europe (Belgium, Norway, Sweden, Switzerland)-SMART 2017-21.

Objectives: To evaluate the in vitro activities of ceftolozane/tazobactam and imipenem/relebactam against clinical isolates of Gram-negative bacilli collected in four central and northern European countries (Belgium, Norway, Sweden, Switzerland) during 2017-21. Methods: Participating clinical laboratories each collected up to 250 consecutive Gram-negative isolates per year from patients with bloodstream, intraabdominal, lower respiratory tract or urinary tract infections. MICs were determined by CLSI broth microdilution and interpreted using 2022 EUCAST breakpoints. ß-Lactamase genes were identified in select ß-lactam-non-susceptible isolate subsets. Results: Ninety-five percent of all Enterobacterales (n = 4158), 95% of ESBL-positive non-carbapenem-resistant Enterobacterales (non-CRE) phenotype Escherichia coli and 85% of ESBL-positive non-CRE phenotype Klebsiella pneumoniae were ceftolozane/tazobactam susceptible. By country, 88% (Belgium), 91% (Sweden, Switzerland) and 96% (Norway) of ESBL-positive non-CRE phenotype Enterobacterales were ceftolozane/tazobactam susceptible. Greater than ninety-nine percent of non-Morganellaceae Enterobacterales and all ESBL-positive non-CRE phenotype Enterobacterales were imipenem/relebactam susceptible. Ceftolozane/tazobactam (96%) and imipenem/relebactam (95%) inhibited most Pseudomonas aeruginosa (n = 823). Both agents retained activity against ≥75% of cefepime-resistant, ceftazidime-resistant and piperacillin/tazobactam-resistant isolates; 56% and 43% of meropenem-resistant isolates were ceftolozane/tazobactam susceptible and imipenem/relebactam susceptible, respectively. By country, 94% (Belgium), 95% (Sweden) and 100% (Norway, Switzerland) of P. aeruginosa were ceftolozane/tazobactam susceptible and 93% (Sweden) to 98% (Norway, Switzerland) were imipenem/relebactam susceptible. Carbapenemase gene carriage among Enterobacterales and P. aeruginosa isolates was generally low (<1%) or completely absent with one exception: an estimated 2.7% of P. aeruginosa isolates from Belgium carried an MBL. Conclusions: Recent clinical isolates of Enterobacterales and P. aeruginosa collected in four central and northern European countries were highly susceptible (≥95%) to ceftolozane/tazobactam and imipenem/relebactam.

Activity of ceftolozane/tazobactam and imipenem/relebactam against clinical gram-negative isolates from Czech Republic, Hungary, and Poland-SMART 2017-2020.

Antimicrobial susceptibility was determined for clinical gram-negative isolates from Czech Republic, Hungary, and Poland, where published data for ceftolozane/tazobactam (C/T) and imipenem/relebactam (IMI/REL) is scarce. C/T was active against 94.3% of Enterobacterales, 10-18% higher than the tested cephalosporins and piperacillin/tazobactam. IMI/REL was the most active tested agent against non-Morganellaceae Enterobacterales (99.7% susceptible). C/T was the most active among all studied agents except colistin against Pseudomonas aeruginosa (96.0% susceptible); susceptibility to IMI/REL was 90.7%. C/T maintained activity against 73.7-85.3% of ß-lactam-resistant or multidrug-resistant P. aeruginosa subsets. C/T and IMI/REL could represent important treatment options for patients from these countries.

Activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa from patients in the Middle East and Africa - Study for Monitoring Antimicrobial Resistance Trends (SMART) 2017-2020.

OBJECTIVES: We evaluated the activity of ceftolozane/tazobactam (C/T), and comparators against clinical Pseudomonas aeruginosa isolates collected for the global Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance program in ten countries in the Middle East and Africa to augment scarce standardized surveillance data in this region. METHODS: Minimum inhibitory concentrations (MICs) were determined using Clinical and Laboratory Standards Institute broth microdilution and interpreted with European Committee on Antimicrobial Susceptibility Testing breakpoints. P. aeruginosa isolates testing with C/T MIC >4 mg/l or imipenem MIC >2 mg/l were screened for ß-lactamase genes. RESULTS: C/T was active against 91.4% and 87.0% of P. aeruginosa isolates from the Middle East and Africa, respectively (14-21 and 7-16 percentage points higher than most ß-lactam comparators, respectively). Considerable variation in susceptibility was seen across countries, which largely correlated with the observed prevalence of carbapenemases and/or extended-spectrum ß-lactamases. Differences across countries were smaller for C/T than for the ß-lactam comparators, ranging from 81% C/T-susceptible among isolates from Jordan to 95% for Qatar. Among subsets resistant to meropenem, ceftazidime, or piperacillin/tazobactam, C/T maintained activity against 51-73% of isolates from the Middle East and against 27-54% from Africa (where metallo-ß-lactamase and GES carbapenemase rates were higher). CONCLUSION: Given the desirability of ß-lactam use among clinicians, C/T represents an important option in the treatment of infections caused by P. aeruginosa.

Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me²⁺ cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

Evaluation of in vitro activity of ceftolozane/tazobactam and comparators against recent clinical bacterial isolates, and genomics of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli isolates that demonstrated resistance to ceftolozane/tazobactam: data from Kuwait and Oman.

Background: The treatment options for infections caused by MDR Gram-negative bacteria have been limited, especially for infections caused by bacteria that produce carbapenemases and/or ESBLs. Ceftolozane/tazobactam is a cephalosporin/ß-lactamase inhibitor developed to treat Gram-negative bacteria. Methods: Ceftolozane/tazobactam and 14 comparators (amikacin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, colistin, ertapenem, imipenem, levofloxacin, meropenem and piperacillin/tazobactam) were evaluated against Pseudomonas aeruginosa and Enterobacterales isolates collected from Kuwait and Oman (n = 606) during 2016-17. In addition, further analysis of resistance mechanisms to ceftolozane/tazobactam was done utilizing WGS. Non-susceptible isolates from ceftolozane/tazobactam surveillance were selected for analysis. Overall, 35 strains underwent WGS. Results: Among isolates from Kuwait, susceptibility of P. aeruginosa, Escherichia coli and Klebsiella pneumoniae to ceftolozane/tazobactam was 79.8%, 95.7% and 87.5%, respectively, and from Oman was 92.3%, 93.1% and 88.5%, respectively. No P. aeruginosa with a ceftolozane/tazobactam MIC <32 mg/L encoded ß-lactamases besides normal chromosomal enzymes (PDC variants or OXA-50-like) whereas all but one P. aeruginosa isolate with MIC >32 mg/L encoded either MBLs (60%), VEB-1 (19%) or additional OXAs (3.7%). Conclusions: Colistin followed by ceftolozane/tazobactam showed the greatest activity against P. aeruginosa. Enterobacterales showed more susceptibility to ceftolozane/tazobactam than to piperacillin/tazobactam, but meropenem and colistin showed better activity.

Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern.

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Collective assessment of antimicrobial susceptibility among the most common Gram-negative respiratory pathogens driving therapy in the ICU.

OBJECTIVES: To describe the pathogen predominance and to evaluate the probability of covering the most common Gram-negative pathogens collectively in both empirical and early adjustment prescribing scenarios in ICU patients with respiratory infections. METHODS: Data were collected from an international cohort of hospitals as part of the SMART Surveillance Program (2018). Susceptibility testing (mg/L) was performed by broth microdilution methods. RESULTS: 7171 Gram-negative respiratory isolates from adult ICU patients across 209 hospitals from 56 different countries were studied. Overall, the most common ICU respiratory pathogens isolated were Pseudomonas aeruginosa (25%), Klebsiella pneumoniae (18%), Acinetobacter baumannii (14%), and Escherichia coli (11%), with inter-regional differences among these pathogens. Among Enterobacterales, 36% were ESBL positive. When the collective susceptibility profile of this set of pathogens (P. aeruginosa plus Enterobacterales; comprising 78% of all organisms isolated) was performed, ceftolozane/tazobactam (84%), followed by meropenem (81%), provided the most reliable in vitro activity in the empirical prescribing scenario compared with other ß-lactam antibiotics. P. aeruginosa co-resistance was common among first-line ß-lactam antibiotics. If P. aeruginosa was non-susceptible to piperacillin/tazobactam, less than one-third were susceptible to meropenem or ceftazidime. In contrast, ceftolozane/tazobactam offered in vitro coverage in over two-thirds of these resistant pathogens. CONCLUSIONS: Ceftolozane/tazobactam demonstrated high cumulative susceptibility levels and in vitro activity in both empirical and adjustment antibiotic prescribing scenarios. High frequency of co-resistance undermines reliable coverage for Gram-negative pathogens already resistant to first-line agents. Ceftolozane/tazobactam would offer additional coverage in this setting.

Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein-protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polß; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer-based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein-protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein-protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein-protein interactions in the coordination of the repair pathway.

Peak Responses during Exercise Treadmill Testing using Individualized Ramp Protocol and Modified Bruce Protocol in Elderly Patients.

BACKGROUND: Exercise capacity is well known to be an important prognostic factor in patients with cardiovascular disease and among healthy persons. AIM: To determine if there are any differences between the peak exercise response during exercise treadmill testing with the individualized ramp protocol and the modified Bruce protocol in elderly patients. MATERIALS AND METHODS: The study included 40 patients (both male and female), aged 70 years and older, who had not had a baseline history of the confirmed coronary artery disease or heart failure diagnoses. All patients underwent exercise treadmill testing using modified Bruce protocol and individualized ramp protocol for 2 consecutive days. Peak heart rate, peak systolic and diastolic blood pressure, peak pressure-rate double product, exercise duration, and peak metabolic equivalents were recorded in both tests. Perceived level of exertion was evaluated using the Borg 10-point scale. RESULTS: The average duration of exercise was longer for the ramp protocol than for the modified Bruce protocol. When the modified Bruce protocol was used, patients achieved a lower workload than they did in using the ramp protocol. The rating of perceived exertion using the revised Borg scale (0 to 10) was 5.6±1.4 for the ramp protocol and 8.7±1.4 for the modified Bruce protocol, which indicates that the patients found the ramp protocol easier. CONCLUSION: In elderly patients the individualized ramp treadmill protocol allows to achieve the optimal test duration with higher degrees of workload and greater patient comfort during the test more often than does the modified Bruce protocol.

A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB.

AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive 'oxyferryl' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein's Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis.

In the repair of the damage to bases, human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme's active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pKa of His-309 located in the active site of the enzyme. This finding revealed that the observed enhancement of enzymatic activity with pH could be associated with deprotonation of not only Tyr-171 but also His-309. The obtained data allowed us to hypothesize that the ionized state of these residues could be a molecular switch between the alternative catalytic mechanisms, which involve different functionalities of these residues throughout the reaction.

The Role of Active-Site Residues Phe98, His239, and Arg243 in DNA Binding and in the Catalysis of Human Uracil-DNA Glycosylase SMUG1.

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.

The impact of single-nucleotide polymorphisms of human apurinic/apyrimidinic endonuclease 1 on specific DNA binding and catalysis.

Human apurinic/apyrimidinic (AP) endonuclease APE1 is a crucial enzyme of the base excision repair (BER) pathway, which is in charge of recognition and initiation of removal of AP-sites in DNA. It is known that some single-nucleotide polymorphism (SNP) variants of APE1 have a reduced activity as compared to wild-type APE1. It has been hypothesized that genetic variation in APE1 might be responsible for an increased risk of some types of cancer. In the present work, analysis of SNPs of the APE1 gene was performed to select the set of variants having substitutions of amino acid residues on the surface of the enzyme globule and in the DNA-binding site, thereby affecting protein-protein interactions or the catalytic reaction, respectively. For seven APE1 variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S), conformational dynamics and catalytic activities were examined. The conformational changes in the molecules of APE1 variants and in a DNA substrate were recorded as fluorescence changes of Trp and 2-aminopurine residues, respectively, using the stopped-flow technique. The results made it possible to determine the kinetic mechanism underlying the interactions of the APE1 variants with DNA substrates, to calculate the rate constants of the elementary stages, and to identify the stages of the process affected by mutation.

Plasma urokinase antigen and C-reactive protein predict angina recurrence after coronary angioplasty.

This study evaluates the predictive value of several biochemical indices of the coagulation-fibrinolysis system, platelet function, and inflammatory state for angina recurrence after successful percutaneous transluminal coronary angioplasty (PTCA). We measured preprocedural and follow-up plasma levels of C-reactive protein (CRP), fibrinogen, and urokinase plasminogen activator antigen (uPA), plasminogen activator inhibitor-1 (PAI-1) activity, tissue plasminogen activator activity, and adenosine diphosphate-induced platelet aggregation in 53 patients with chronic stable angina who underwent successful elective PTCA of single hemodynamically significant lesions in coronary arteries. All patients were followed up for 12 months after PTCA. The Cox proportional hazards model was used to assess the association of variables with angina recurrence rate. At the end of the follow-up, 16 patients had angina recurrence. Among 36 clinical, biochemical, and angiographic variables, the duration of stable angina more than 12 months before PTCA (χ (2) = 5.73; P = 0.02, hazard ratio (HR) 3.7, 95 % confidence interval (CI) 1.26-10.6), high baseline levels of CRP (>7 mg/l) (χ (2) = 8.34; P = 0.004, HR 2.9, 95 % CI 1.4-5.9), uPA antigen baseline (>1 ng/ml) (χ (2) = 17.11; P = 0.0001, HR 11.5, 95 % CI 3.6-36.7) and 48 h after PTCA (χ (2) = 15.73; P = 0.0001, HR 8.8, 95 % CI 3.01-25.96), baseline PAI-1 activity (>18 IU/ml) (χ (2) = 9.37; P = 0.002, HR 7.6, 95 % CI 2.07-27.84) were significant predictors of recurrent angina by univariate analyses. According to stepwise multivariate analyses, only the levels of plasma uPA antigen and serum CRP were shown to be significant independent predictors of angina recurrence (multivariate uPA χ (2) = 8.22, P = 0.004, HR 6.2, 95 % CI 1.78-21.67; CRP χ (2) = 4.09, P = 0.04, HR 1.9, 95 % CI 1.02-3.68). High preprocedural plasma uPA and serum CRP levels are indicative of angina recurrence after successful PTCA, and are valuable for the prognosis of restenosis.