55. Impact of Testing Methodology and Reporting on Time to Preferred Antibiotic Therapy in Extended Spectrum Beta-Lactamase producing Enterobacteriaceae (ESBL-E) Bloodstream Infections

Background Harborview Medical Center (HMC) identifies organisms and an ESBL genotype (CTX-M) via Verigene® Gram-Negative Blood Culture Nucleic Acid Test (BC-GN). University of Washington-Montlake (UWML) uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for organism identification directly from positive blood cultures and ceftriaxone results by Kirby Bauer disk diffusion (KB) are reported 18 hours later. No ESBL comment is reported at UWML. We aimed to determine whether the methodology in identification and reporting of ESBL-E from blood cultures between two hospitals has an impact on time to preferred therapy with a carbapenem antibiotic. Methods Retrospective observational study conducted at UWML and HMC in Seattle, WA between 1/10/2015 and 9/15/2020. Adult patients were eligible if they had ≥1 positive blood culture with an Enterobacteriaceae isolate resistant to ceftriaxone and were on antibiotic treatment. The primary outcome was the difference in time to preferred definitive therapy with a carbapenem antibiotic in patients an ESBL-E bloodstream infection (BSI) identified by Verigene® vs. MALDI-TOF MS/KB. Results A total of 199 patients were screened; 67 were included for UWML and 68 at HMC. The average time to initiation of a carbapenem antibiotic was 42 ±26.5 hours at UWML and 28 ±19.7 hours at HMC. A t-test detected a difference in time to preferred therapy between a Verigene® vs. MALDI-TOF MS/KB tested ESBL-E BSI [95% confidence interval (CI), 5.3-22.9]. The hazard ratio to carbapenem initiation for HMC is 1.73643 [95% CI, 1.1405-2.644]. Conclusion A statistically significant difference in time to preferred definitive therapy among patients with an ESBL-E BSI processed by Verigene® was found compared to MALDI-TOF MS. The results suggest standardization in protocols between the UWML and HMC hospitals is warranted. Disclosures Andrew Bryan, MD, PhD, Shionogi Inc. (Grant/Research Support)

CARBAPENEM ANTIBIOTIC DOSE MODE FOR PEDIATRIC PATIENT AT CAN THO CHILDREN’S HOSPITAL

Objective: Analyzing the dose regimen of carbapenem antibiotics used in pediatric patients at Can Tho Children’s Hospital.Objects and methods: retrospective-descriptive study on 140 medical records of patients being treated at departments of Can Tho Children’s Hospital during the period from June 1st, 2020 toDecember 31st, 2020. Results: Regarding the dosage regimen characteristics of meropenem showed that 20.3% and 10.8%of meropenem’s indications for the dose of meropenem for non-central nervous system infections and meningitis treament was appropriate according to the recommendations. Meanwhile, up to63.5% of the dose was higher than the recommended dose and 5.5% of the treatment dose was lower than the recommended dose. Regarding imipenem dosage regimen characteristics from the resultsshowed that 71.2% of the dose of imipenem for the treatment of infections non-central nervous system complied with recommendations according to the literature. However, more than 20% of the recommended dose for patients was lower than recommended dose. Conclusion: The meropenem dose regimen was consistent with the recommendation was low. The imipenem dose regimen was consistent with the relatively high recommendation.

Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme

Cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes acting during carbapenem antibiotic biosynthesis carry out radical-mediated methyl transfers that underlie the therapeutic usefulness of these essential medicines. Here we present x-ray crystal structures of TokK, which are representative of this functional class, containing its two metallocofactors and determined in the presence and absence of carbapenam substrate. The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggests that substrate positioning governs the rate of each methylation event.

SITUATION USING CARBAPENEM

Objective: To survey the use of carbapenem antibiotics at An Sinh General Hospital. Subjects and methods: retrospective descriptive study on 100 medical records of patients being treated in departments of An Sinh General Hospital from June 1st, 2020 to December 31st, 2020. Results: The carbapenem antibiotic used at An Sinh General Hospital mainly indicated for the treatment of pneumonia accounted for the highest percentage with 53.3%. There were 100% of strains of Haemophilus influenzae; Rhizobium radiobacter; Proteus. cp; Streptococcus mitis, which was also sensitive to carbapenem. 100% strains of Stenotrophomonas maltophilia bacteria was resistant to meropenem; 01 strain of Burkhorderia vietnamiensis was isolated against imipenem; 2 of 3 strains of Staphylococcus aereus isolated were resistant to meropenem. Carbapenem was mainly used in combination regimens. The proportion of the combination in the initial regimen was 100%. The rate of combination of 3 antibiotics was 22.7%. In the replaceable regimen, the combination rate was 82.2%. Conclusion: The carbapenem group was indicated for the treatment of pneumonia. Strains of the bacteria Haemophilus influenzae; Rhizobium radiobacter; Proteus. cp; Streptococcus mitis was also sensitive to carbapenem. Bacteria strains Stenotrophomonas maltophilia, Burkhorderia vietnamiensis and Staphylococcus aereus were resistant to meropenem. Carbapenem was mainly used in combination regimens.

The value of serum procalcitonin in the anti-infection therapy of acute stroke patients

Objectives: To investigate the value of dynamic monitoring of serum procalcitonin (PCT) in anti-infective therapy of patients with acute stroke. Methods: This is a case control retrospective study of acute stroke patients conducted from July 2016 to October 2018, in the Department of Neurology, Affiliated Hospital of Hebei University, who who reached within twenty four hours. They, were selected as the study subjects who were divided into infection group and non-infection group according to the inclusion and exclusion criteria. The serum PCT and CRP levels were compared between the two groups at 24 hours, 48 hours and 72 hours. In order to judge the changes of PCT level and the infection of stroke patients, different kinds of antibiotics were used for corresponding treatment. Retrospective analysis of the cases that did not monitor PCT anti infective treatment before July 2016 were compared with the cases that monitored PCT to guide anti infective treatment after July 2016, and compared the efficacy of antibiotics. Results: The serum PCT level of patients in the infection group was significantly higher than that of patients in the noninfection group (P<0.001). For the patients whose PCT<0.5 ng/ml within 72 hour, anti-infective therapy was not administered. However, for those patients whose PCT<0.5 ng/ml and CRP rose significantly, WBC, body temperature and chest CT were closely monitored. For the patients whose PCT increased slightly (0.5 ng/ml<PCT<2.0 ng/ml), first-generation and second-generation cephalosporin or semisynthetic penicillin, such as mezlocillin, were administered. For the patients whose PCT increased moderately (5 ng/ml>PCT>2 ng/ml), mezlocillin/ sulbactam or ceftriaxone/ tazobactam was administered. For patients whose PCT increased significantly (PCT>5 ng/ml), carbapenem antibiotic or a combination of two antibiotics was administered. Conclusion: Dynamic detection of serum PCT concentration can make accurate judgment on the severity of bacterial infection in patients with acute stroke and guide the rational application of antibiotics. doi: https://doi.org/10.12669/pjms.37.4.3932 How to cite this:Wang H, Li Y, Li X, Wang Z. The value of serum procalcitonin in the anti-infection therapy of acute stroke patients. Pak J Med Sci. 2021;37(4):---------. doi: https://doi.org/10.12669/pjms.37.4.3932 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Meropenem Administered via Intravenous Regional Limb Perfusion for Orthopedic Sepsis in Horses: A Clinical Retrospective Study

Septic synovitis is a critical orthopedic condition in horses. Early intervention is key, with antibiotic therapy typically initiated prior to culture and susceptibility reports becoming available. The pharmacokinetics of several antibiotics have been studied in horses for use in intravenous regional limb perfusion (IVRLP) for septic synovitis, including the carbapenem antibiotic, meropenem. For a variety of factors, some veterinary clinicians may select IVRLP meropenem as therapy for these cases. Meropenem is a vital antibiotic in human medicine, making veterinary use divisive. However, verifying the efficacy of meropenem contrasted to other IVRLP antibiotics is essential for appropriate antimicrobial stewardship. To investigate this, equine patient medical records at a single veterinary teaching hospital were examined. Cases treated with meropenem or gentamicin via IVRLP for septic synovitis were retrospectively analyzed for demographics, diagnostics, treatments, outcomes, and adverse effects. Twenty-three meropenem and 37 gentamicin treated horses were analyzed; demographic information was similar between groups. In the meropenem group, nine horses received meropenem only; the remainder received another antibiotic initially then changed to meropenem. Structures infected included joints (meropenem = 13, gentamicin = 17), tendon sheaths (meropenem = 5, gentamicin = 8) and navicular bursae (meropenem = 2, gentamicin = 6). Overall survival to discharge was 86% (52/60), with meropenem 91% (21/23) and gentamicin 84% (31/37), with no statistically significant differences noted between meropenem or gentamicin groups for overall survival to discharge or outcome after discharge. Twenty-four of 26 bacterial isolates obtained from culture were reported as sensitive to imipenem, a carbapenem antibiotic similar to meropenem. Reported susceptibility to other antibiotics such as ceftiofur (n = 22/26), ampicillin (n = 18/26), amikacin (n = 15/26), or gentamicin (n = 12/26) was also frequently present. In the population of this study, antimicrobial activity augmented with IVRLP using either meropenem or gentamicin both appear to be an effective treatment for septic synovial structures, therefore, less critical antimicrobials may be a viable and more judicious treatment option.

Proposed Guidelines for the Management of ESBL in Prosthetic Joint Infections

The aim of this paper is to establish guidelines for the management of extendedspectrum beta-lactamases (ESBL) associated prosthetic joint infections (PJI). This study reviewed 21 patients in the literature documented with ESBL associated PJI. Literature suggests that patients with ESBL PJI are stratified into either early infections (<3 weeks) or late infections (>3 weeks), for which, appropriate laboratory and imaging studies need to be completed. Favorable outcomes require a two-stage revision with an antibiotic-impregnated spacer and a prolonged course of intravenous carbapenem antibiotic.

KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

Mechanistic Investigations of Metallo-β-Lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC₅₀) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn²⁺, Ca²⁺ and Mg²⁺. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing <i>E. coli</i> strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate <b>12</b> proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC₅₀ values in the low nanomolar range against both enzymes. Interestingly, while compound <b>12</b> was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.

Mechanistic Investigations of Metallo-β-Lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC₅₀) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn²⁺, Ca²⁺ and Mg²⁺. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing <i>E. coli</i> strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate <b>12</b> proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC₅₀ values in the low nanomolar range against both enzymes. Interestingly, while compound <b>12</b> was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.