Gram-negative Activity Research Articles

Allenamides as a Powerful Tool to Incorporate Diversity: Thia-Michael Lipidation of Semisynthetic Peptides and Access to β-Keto Amides.

Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semisynthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22 lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a β-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.

Multidrug efflux in Gram-negative bacteria: structural modifications in active compounds leading to efflux pump avoidance

Gram-negative bacteria cause the majority of critically drug-resistant infections, necessitating the rapid development of new drugs with Gram-negative activity. However, drug design is hampered by the low permeability of the Gram-negative cell envelope and the function of drug efflux pumps, which extrude foreign molecules from the cell. A better understanding of the molecular determinants of compound recognition by efflux pumps is, therefore, essential. Here, we quantitatively analysed the activity of 73,737 compounds, recorded in the publicly accessible database CO-ADD, across three strains of E. coli – the wild-type, the efflux-deficient tolC variant, and the hyper-permeable lpxC variant, to elucidate the molecular principles of evading efflux pumps. We computationally investigated molecular features within this dataset that promote, or reduce, the propensity of being recognised by the TolC-dependent efflux systems in E. coli. Our results show that, alongside a range of physicochemical features, the presence or absence of specific chemical groups in the compounds substantially increases the probability of avoiding efflux. A comparison of our findings with inward permeability data further underscores the primary role of efflux in determining drug bioactivity in Gram-negative bacteria.

Green Synthesis and Characterization of Hydrozincite Nanoparticles from Wild Passion Fruit

Abstract: Green synthesis of nano particles involves the use of whole plant or parts of plant for the bioreduction of metal ions into their elemental form. Hydrozincite nano particles were synthesized using the wild passion fruit. The fruit has got so many health benefits. The synthesized nano particles were characterized by X-Ray diffraction(XRD), Fourier Transform Infra-Red spectroscopy(FT-IR), UV-Vis absorption spectroscopy(UV), Scanning Electron Microscope(SEM) and Energy Dispersive X-Ray Diffraction(EDX). Using XRD the average particle size was found to be 14.032nm. The functional groups of the prepared sample were identified using FT-IR analysis. From UV the absorption peak was noticed at 302.199 nm which confirms the particle was in nano range. The surface morphology of the synthesized particles was noticied as bread spongy from the SEM analysis. The chemical compounds in the nano particles were identified using EDX analysis. Anti-microbial resistance is one of the greatest challenges to global health. The prepared nano particle shows a good effect for both gram-positive and gramnegative anti-bacterial activity.

Approaches to Testing Novel β-Lactam and β-Lactam Combination Agents in the Clinical Laboratory.

The rapid emergence of multi-drug resistant Gram-negative pathogens has driven the introduction of novel β-lactam combination agents (BLCs) to the antibiotic market: ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, cefiderocol, and sulbactam-durlobactam. These agents are equipped with innovative mechanisms that confer broad Gram-negative activity, notably against certain challenging carbapenemases. While their introduction offers a beacon of hope, clinical microbiology laboratories must navigate the complexities of susceptibility testing for these agents due to their diverse activity profiles against specific β-lactamases and the possibility of acquired resistance mechanisms in some bacterial isolates. This review explores the complexities of these novel antimicrobial agents detailing the intricacies of their application, providing guidance on the nuances of susceptibility testing, interpretation, and result reporting in clinical microbiology laboratories.

1274. Long-term Sustainability of Improvements in Antibiotic Prescribing for Inpatient Skin and Soft Tissue Infections after Implementation of Local Treatment Guidelines

Abstract Background Institutional guidelines for the management of inpatient skin and soft tissue infections (SSTIs) were implemented at an academic public safety-net hospital in 2009. This led to an immediate and marked decrease in unnecessary use of broad-spectrum antibiotics and durations of therapy. The objective of this study was to determine if these changes in prescribing have been sustained over time. Methods This was a retrospective review of patients hospitalized with cellulitis or abscess at Denver Health Medical Center between July 2016 and August 2022 (maintenance period). Case identification and data collection methods were identical to those used in a previous publication describing pre-intervention (January to December 2007) and intervention (July 2009 to July 2010) period data. The main outcomes were the proportion of patients exposed to an antibiotic with broad-spectrum gram-negative or antipseudomonal activity and the median duration of therapy. Results A total of 186 patients from the maintenance period were included for comparison with those from the pre-intervention (n = 169) and intervention (n = 175) periods. In the maintenance period, the median patient age was 41 years and 74% were male. The most common comorbid condition was injection drug use (27%). Overall clinical characteristics were similar between the three periods. The proportion of patients exposed to an antibiotic with broad gram-negative activity was 66% during the pre-intervention period, 33% during the intervention period, and 27% during the maintenance period. The proportion of patients exposed to an antibiotic with antipseudomonal activity was 28%, 18%, and 16% during the pre-intervention, intervention, and maintenance periods, respectively. The median total duration of therapy was 13 (IQR 10-15), 10 (IQR 9-12), and 8 (IQR 7-10) days in the pre-intervention, intervention, and maintenance periods, respectively. Conclusion Reductions in use of broad-spectrum antibiotics and durations of therapy for inpatients with SSTIs were sustained 12 years after implementation of a local treatment guideline. These findings add to the evidence that local guidelines are an effective antibiotic stewardship tool and demonstrate that resultant improvements in prescribing can be sustained over prolonged periods. Disclosures Timothy C. Jenkins, MD, Basilea: Clinical events adjudication committee

Antibacterial activity of Ag-graphene nanocomposites against Gram-Positive and Gram-negative bacteria

Background: Many pathogenic microorganisms, including bacteria, viruses, and fungi, which are common sources of disease and infection in both humans and animals, have a significant impact on human health. To combat these microorganisms, scientists and technicians are steadily attempting to develop novel and potent antimicrobial agents. Recently, graphene nanosheets (GNs) based nanocomposites (NCs) have shown promising potential as antibacterial activity against microorganisms. The present is an attempt to examine the antimicrobial effect of Silver (Ag)/GNs NCs against gram-positive (Bacillus thuringiensis, Staphylococcus aureus, and Enterococcus faecalis) and gram-negative (Salmonella typhi) bacteria. Methods: In this study, Ag/GNs NCs have been synthesized by the solvothermal method. X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), UV-vis spectroscopy, field emission scanning electron microscopy (FESEM), and X-ray photoelectron spectroscopy have all been used to study the Ag/GNs NCs. The antibacterial activity of synthesized GO and Ag/GNs NCs was evaluated against microorganisms using the disk diffusion method. Results: The elemental analysis of synthesized nanomaterial revealed that GO and Ag ions have been reduced by citric acid, and led to the successful formation of Ag/GNs NCs. The resultant NCs have been examined for their antibacterial activity against gram-positive (Bacillus thuringiensis, Staphylococcus aureus, and Enterococcus faecalis) and gram-negative (Salmonella typhi) bacteria. It was observed that Ag/GNs NCs markedly inhibit gram-positive and gram-negative bacteria. Conclusion: The prepared Ag/GNs NCs have the potential for long-term gram-positive and gram-negative bacteria-targeting antibacterial activities and grasp the ability in combating public health threats.

Successful Use of Cefiderocol to Treat a Multidrug-resistant Stenotrophomonas maltophilia Ventilator-associated Pneumonia in an Extremely Preterm Neonate.

Ventilator-associated pneumonia (VAP) caries a morbidity and mortality risk in the preterm neonate, particularly in the context of rising global antimicrobial resistance driving infections due to multidrug-resistant Gram-negative bacteria. Cefiderocol, a siderophilic cephalosporin, has broad Gram-negative antimicrobial activity and central nervous system penetration and is used for the treatment of hospital-acquired pneumonia or VAP in adults. Scarce data exists on its use in neonates. A female neonate born at 26 + 6 weeks developed VAP at 21 days of life. She was commenced on corticosteroids, vancomycin and ceftazidime but continued to deteriorate. Sputum cultures yielded Stenotrophomonas maltophilia resistant to trimethoprim/sulfamethoxazole, ciprofloxacin and ceftazidime, with potential susceptibility to cefiderocol. Cerebrospinal fluid showed an elevated white cell count. In view of worsening respiratory and hemodynamic status, antibiotic treatment was changed to cefiderocol monotherapy at 30 mg/kg/dose every 8 hours. Within 72 hours of commencing cefiderocol, the neonate was successfully extubated to variable-flow continuous positive airway pressure and showed ongoing clinical improvement. Cefiderocol was integral for the care of our neonate without any immediate adverse safety consequences. We relied on dosing data from a conference abstract, due to the paucity of evidence on the use of novel antimicrobials. This lack of evidence is particularly concerning given preterm neonates are particularly vulnerable to infections with multidrug-resistant Gram-negative organisms due to their immature immune systems, prolonged hospital stay, repeated interventions and antimicrobial exposure.

Valorization of mangrove leaves (Sonneratia alba) based on leaf age as a functional salt preparation

Mangrove leaves (Sonneratia alba) have bioactive components that have the potential as natural antioxidants, NaCl salts, macro and micro minerals, and gram-positive and gram-negative antibacterial activities. However, mangrove leaves are still not fully utilized, so it is necessary to valorize to increase the product's added value. This study aimed to characterize S. alba mangrove leaves as a functional salt preparation based on leaf age. Valorization of mangrove leaves was carried out through an extraction method using distilled water with test parameters consisting of yield, mineral analysis, Na:K ratio, NaCl analysis, antioxidant activity, and organoleptic tests. The salt characteristics of young S. alba mangrove leaves include a yield of 6.67%; tastes salty followed by a bitter taste; antioxidant activity IC50 of 14.34 ​g/mL; dominant minerals of 5.632% sodium and 4.542% potassium; ratio Na:K 1.24; and 22.88% NaCl levels. While the salt characteristics of the old S. alba mangrove leaves include a yield of 6.37%; tastes salty followed by a bitter taste; antioxidant activity IC50 of 21.12 ​g/mL; dominant minerals of 7.686% sodium and 3.294% potassium; ratio Na:K 2.33; and 27.01% NaCl content. Both young and old mangrove leaf salts fulfilled the dietary salt standards.

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

The antibacterial and cytocompatibility of the polyurethane nanofibrous scaffold containing curcumin for wound healing applications

Abstract In today’s world, wound healing is a growing serious problem for clinical institutes. In this study, nanofibrous scaffolds were prepared using polyurethane as a mat scaffold. Also, by immersing curcumin as an antibacterial component another scaffold was fabricated using the electrospinning technique. The obtained scaffolds were characterized by means of scanning electron microscopy, tensile analysis, porosity, and water vapor transmission rate. MTT and DAPI staining were used to prove the biocompatibility and cell attachment of the nanofibers. The curcumin incorporated into the PU scaffold can stop both the Gram-negative and Gram-positive bacteria activities through direct contact with them. Studies showed that the PU/Curcumin scaffold has considerable ability to play a key role in wound healing applications.

New sustainable antimicrobial chitosan hydrogels based on sulfonamides and its nanocomposites: Fabrication and characterization

Chitosan (Ch), a linear cationic biopolymer, has broad medical applications. In this paper, new sustainable hydrogels (Ch-3, Ch-5a, Ch-5b) based on chitosan/sulfonamide derivatives 2-chloro-N-(4-sulfamoylphenethyl) acetamide (3) and/or 5-[(4-sulfamoylphenethyl) carbamoyl] isobenzofuran-1,3-dione (5) were prepared. Hydrogels (Ch-3, Ch-5a, Ch-5b) were loaded (Au, Ag, ZnO) NPs to form its nanocomposites to improve the antimicrobial efficacy of chitosan. The structures of hydrogels and its nanocomposites were characterized using different tools. All hydrogels displayed irregular surface morphology in SEM, however hydrogel (Ch-5a) revealed the highest crystallinity. The highest thermal stability was shown by hydrogel (Ch-5b) compared to chitosan. The nanocomposites represented nanoparticle sizes <100 nm. Antimicrobial activity was assayed for hydrogels using disc diffusion method exhibited great inhibition growth of bacteria compared to chitosan against S. aureus, B. subtilis and S. epidermidis as Gram-positive, E. coli, Proteus, and K. pneumonia as Gram-negative and antifungal activity against Aspergillus Niger and Candida. Hydrogel (Ch-5b) and nanocomposite hydrogel (Ch-3/Ag NPs) showed higher colony forming unit (CFU) and reduction% against S. aureus and E. coli reaching 97.96 % and 89.50 % respectively in comparison with 74.56 % and 40.30 % for chitosan respectively. Overall, fabricated hydrogels and its nanocomposites enhanced the biological activity of chitosan and it can be potential candidates as antimicrobial drugs.

RND Pump-Mediated Efflux of Amotosalen, a Compound Used in Pathogen Inactivation Technology to Enhance Safety of Blood Transfusion Products, May Compromise Its Gram-Negative Anti-Bacterial Activity.

Pathogen inactivation is a strategy to improve the safety of transfusion products. The only pathogen reduction technology for blood products currently approved in the US utilizes a psoralen compound, called amotosalen, in combination with UVA light to inactivate bacteria, viruses, and protozoa. Psoralens have structural similarity to bacterial multidrug efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through genetic, biophysical, and molecular modeling analysis. The main efflux systems in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa are tripartite resistance-nodulation-cell division (RND) systems, which span the inner and outer membranes of Gram-negative pathogens, and expel antibiotics from the bacterial cytoplasm into the extracellular space. We provide evidence that amotosalen is an efflux substrate for the E. coli AcrAB, Acinetobacter baumannii AdeABC, and P. aeruginosa MexXY RND efflux pumps. Furthermore, we show that the MICs for contemporary Gram-negative bacterial isolates for these species and others in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens. IMPORTANCE Pathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by major efflux pumps in E. coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the MICs for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to identify possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

Transforming Cross-Linked Cyclic Dimers of KR-12 into Stable and Potent Antimicrobial Drug Leads.

Is it possible to enhance structural stability and biological activity of KR-12, a truncated antimicrobial peptide derived from the human host defense peptide LL-37? Based on the mapping of essential residues in KR-12, we have designed backbone-cyclized dimers, cross-linked via a disulfide bond to improve peptide stability, while at the same time improving on-target activity. Circular dichroism showed that each of the dimers adopts a primarily alpha-helical conformation (55% helical content) when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new cross-linked cyclic form. Compared to KR-12, one of the cross-linked dimers showed 16-fold more potent antimicrobial activity against human pathogens Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans and 8-fold increased activity against Escherichia coli. Furthermore, these peptides retained antimicrobial activity at physiologically relevant conditions, including in the presence of salts and in human serum, and with selective Gram-negative antibacterial activity in rich growth media. In addition to giving further insight into the structure-activity relationship of KR-12, the current work demonstrates that by combining peptide stabilization strategies (dimerization, backbone cyclization, and cross-linking via a disulfide bond), KR-12 can be engineered into a potent antimicrobial peptide drug lead with potential utility in a therapeutic context.

1833. Evaluating the Impact of Reported Beta-Lactam Allergies on Clinical Outcomes in Gram-Negative Bloodstream Infections: A Retrospective Cohort Study

Abstract Background Approximately 15% of hospitalized patients are labeled as beta-lactam (BL) allergic. Despite this, it is estimated that &amp;gt; 90% of patients do not have clinically significant hypersensitivity reactions and are BL-tolerant. The BL allergy label is associated with a host of downstream consequences, including receipt of suboptimal antibiotic therapy, healthcare-associated infections, and bacterial resistance. There are limited data investigating the impact of this allergy label on clinical outcomes in bloodstream infections caused by Gram-negative pathogens (GN-BSI). Methods This was a retrospective, single center, cohort study evaluating the impact of BL allergies on clinical outcomes in patients with GN-BSI from January 2017 to August 2021. Adult inpatients with index blood cultures positive for Pseudomonas species or Enterobacterales who received intravenous antibiotics with Gram-negative activity were included. The primary endpoint was time to effective therapy (defined as treatment with in vitro activity against a causative pathogen, as determined by phenotypic susceptibilities) and the key secondary endpoint was 30-day all-cause mortality. Results Overall, 120 patients were included, representing 30 BL-allergic (BL-A) and 90 BL-non-allergic (BL-NA) patients. Blood cultures positive for Pseudomonas species or Enterobacterales were evenly distributed (Table 1). Among Enterobacterales, Escherichia coli was implicated in 25% of cases overall. The most common source of infection was intra-abdominal infection. Across groups, median quick Pitt Bacteremia Score was 1 (IQR 0-2). Infectious Diseases was consulted in 37% of BL-A patients vs. 29% of BL-NA patients. Of BL allergies reported, 86% were classified as likely immunoglobulin E (IgE) mediated reactions. Allergy was not consulted in any of these cases. Median time to effective therapy was 162 minutes (IQR 20-824 minutes) vs. 103 minutes (IQR 27-775 minutes) in the BL-A and BL-NA groups, respectively. Thirty-day mortality occurred in 31% of BL-A and 21% of BL-NA patients. Conclusion Increased time to effective therapy and mortality rates were observed among patients with reported BL allergies. These findings highlight the importance of allergy clarification in antibiotic stewardship. Disclosures Kathleen Degnan, MD, Gilead: Grant/Research Support.

1839. Impact of Hypoalbuminemia on Ceftriaxone Treatment Failure in Patients With Enterobacterales Bacteremia

Abstract Background Ceftriaxone (CRO) is a third-generation cephalosporin that is commonly prescribed due to its robust Gram-negative activity and lack of renal dose adjustments. It is also highly protein bound allowing for once daily dosing. In patients with hypoalbuminemia, however, the proportion of free drug is increased thus increasing the rate at which CRO is renally eliminated. This increased clearance could lead to lower serum concentrations and increased risk of treatment failure. Methods We performed a retrospective, cohort study to assess the impact of hypoalbuminemia (serum albumin ≤ 2.5 g/dL) on treatment failure among patients with monomicrobial Enterobacterales bacteremia. Adult patients who received &amp;gt; 72 hours of CRO therapy for susceptible Enterobacterales bacteremia between May 1, 2016 and April 30, 2021 were eligible for inclusion. The primary outcome of treatment failure was a composite of inpatient mortality or escalation to an intravenous anti-pseudomonal antibiotic or ertapenem. Secondary outcomes included hospital length of stay, total duration of antibiotic therapy, and time to infection resolution. Baseline characteristics and outcomes were compared using R Studio (Version 4.1.0) in a 1:1 propensity-matched cohort. Results After propensity score matching, 142 patients were included in each study group. The most common organisms in our cohort were Escherichia coli (71.5%), Klebsiella pneumoniae (14.1%), and Proteus mirabilis (5.3%). The most frequently implicated source of infection was the urinary tract (71.5%). Interestingly, treatment failure was numerically higher in the hypoalbuminemia group but was not statistically significant (12.0% vs. 7.7%, p = 0.23). All secondary outcomes were similar between groups. Among the subgroup of patients admitted to the ICU at baseline, the difference in treatment failure was even greater between patients with hypoalbuminemia and those without (23.3% vs. 7.5%, P = 0.07). Conclusion Hypoalbuminemia did not appear to have a significant impact on clinical outcomes among patients with Enterobacterales bacteremia treated with CRO. However, critically ill patients may be subject to higher incidence of treatment failure in the presence of hypoalbuminemia. Disclosures All Authors: No reported disclosures.

Covalent DNA Binding Is Essential for Gram-Negative Antibacterial Activity of Broad Spectrum Pyrrolobenzodiazepines.

It is urgent to find new antibiotic classes against multidrug-resistant bacteria as the rate of discovery of new classes of antibiotics has been very slow in the last 50 years. Recently, pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic-heterocycle have been identified as a new broad-spectrum antibiotic class with activity against Gram-negative bacteria. The active imine moiety of the reported lead pyrrolobenzodiazepine compounds was replaced with amide to obtain the non-DNA binding and noncytotoxic dilactam analogues to understand the structure-activity relationship further and improve the safety potential of this class. The synthesised compounds were tested against panels of multidrug-resistant Gram-positive and Gram-negative bacteria, including WHO priority pathogens. Minimum inhibitory concentrations for the dilactam analogues ranged from 4 to 32 mg/L for MDR Gram-positive bacteria, compared to 0.03 to 2 mg/L for the corresponding imine analogues. At the same time, they were found to be inactive against MDR Gram-negative bacteria, with a MIC &gt; 32 mg/L, compared to a MIC of 0.5 to 32 mg/L for imine analogues. A molecular modelling study suggests that the lack of imine functionality also affects the interaction of PBDs with DNA gyrase. This study suggests that the presence of N10-C11 imine moiety is crucial for the broad-spectrum activity of pyrrolobenzodiazepines.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Low Release Study of Cefotaxime by Functionalized Mesoporous Silica Nanomaterials

As a third-generation β-lactam antibiotic, cefotaxime shows a broad-spectrum with Gram-positive and Gram-negative bacteria activity and is included in WHO's essential drug list. In order to obtain new materials with sustained release properties, the present research focuses on the study of cefotaxime absorption and desorption from different functionalized mesoporous silica supports. The MCM-41-type nanostructured mesoporous silica support was synthesized by sol-gel technique using a tetraethyl orthosilicate (TEOS) route and cetyltrimethylammonium bromide (CTAB) as a surfactant, at room temperature and normal pressure. The obtained mesoporous material (MCM-41 class) was characterized through nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), N2 absorption-desorption (BET) and Fourier transform infrared spectroscopy (FT-IR), proving a good micro-structured homogeneity (SEM images), a high surface area (BET, 1029 m2/g) correlated with high silanolic activity (Q3/Q4 peak ratio from 29Si MAS-NMR), and an expected uniform hexagonal structure (2-3 nm, HRTEM). In order to non-destructively link the antibiotic compound on the solid phase, MCM-41 was further functionalized in two steps: with aminopropyl trimethoxysilane (APTMS) and glutaraldehyde (GA). Three cefotaxime-loaded materials were comparatively studied for low release capacity: the reference material with adsorbed cefotaxime on MCM-41, MCM-41/APS (aminopropyl silyl surface functionalization) adsorbed cefotaxime material, and APTMS-GA bounded MCM-41-cefotaxime material. The slow-release profiles were obtained by using an on-flow modified HPLC system. A significant improved release capacity was identified in the case of MCM-41/APS/GA-cefotaxime due to the covalent surface grafting of the biological active compound, recommending this class of materials as an effective carrier of bioactive compounds in wound dressing, anti-biofilm coatings, advanced drugs, and other related applications.

A Short History of Topoisomerases at Actelion Pharmaceuticals.

The discovery of novel antibacterials devoid of cross resistance is of utmost importance. At the same time, biological pathways and processes suitable to be targeted are limited. At Actelion Pharmaceuticals we decided to work on novel bacterial topoisomerase inhibitors (NBTI) to discover new antibiotics with broad spectrum activity and limited resistance development for use against severe hospital infections. This paper summarizes the learnings and results of our efforts in the field, which led to the discovery of multiple chemical classes with potent Gram-negative activity and ultimately to the selection of several compounds that underwent preclinical profiling.

The Ins and Outs of Susceptibility Testing for New β-Lactam/β-Lactamase Inhibitor Combinations for Gram-Negative Organisms.

Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam are among the newest β-lactam/β-lactamase inhibitors (BL/BLIs) introduced to the North American antibiotic market. All have broad Gram-negative activity, including against certain carbapenemases. Despite this, susceptibility testing is warranted due to variable activity against certain β-lactamases (e.g., oxacillinases) and the presence of acquired resistance mechanisms in some isolates. Here, we discuss what we know about these new antimicrobial agents and how to navigate implementation of susceptibility testing and reporting of these agents in clinical laboratories.