Sulbactam-durlobactam: A Step Forward in Treating Carbapenem-Resistant Acinetobacter baumannii (CRAB) Infections.

Antimicrobial resistance in gram-negative pathogens, such as Acinetobacter baumannii, is a serious threat to human health. Sulbactam-durlobactam, a unique ß-lactam and a ß-lactamase inhibitor combination, is a novel agent targeted against carbapenem-resistant A. baumannii. This supplement provides a summary of the development of SUL-DUR, discussing its unique features and role in treating infections caused by CRAB pathogens.

The Pharmacokinetics/Pharmacodynamic Relationship of Durlobactam in Combination With Sulbactam in In Vitro and In Vivo Infection Model Systems Versus Acinetobacter baumannii-calcoaceticus Complex.

Sulbactam-durlobactam is a ß-lactam/ß-lactamase inhibitor combination currently in development for the treatment of infections caused by Acinetobacter, including multidrug-resistant (MDR) isolates. Although sulbactam is a ß-lactamase inhibitor of a subset of Ambler class A enzymes, it also demonstrates intrinsic antibacterial activity against a limited number of bacterial species, including Acinetobacter, and has been used effectively in the treatment of susceptible Acinetobacter-associated infections. Increasing prevalence of ß-lactamase-mediated resistance, however, has eroded the effectiveness of sulbactam in the treatment of this pathogen. Durlobactam is a rationally designed ß-lactamase inhibitor within the diazabicyclooctane (DBO) class. The compound demonstrates a broad spectrum of inhibition of serine ß-lactamase activity with particularly potent activity against class D enzymes, an attribute which differentiates it from other DBO inhibitors. When combined with sulbactam, durlobactam effectively restores the susceptibility of resistant isolates through ß-lactamase inhibition. The present review describes the pharmacokinetic/pharmacodynamic (PK/PD) relationship associated with the activity of sulbactam and durlobactam established in nonclinical infection models with MDR Acinetobacter baumannii isolates. This information aids in the determination of PK/PD targets for efficacy, which can be used to forecast efficacious dose regimens of the combination in humans.

Global Epidemiology and Mechanisms of Resistance of Acinetobacter baumannii-calcoaceticus Complex.

Acinetobacter baumannii-calcoaceticus complex is the most commonly identified species in the genus Acinetobacter and it accounts for a large percentage of nosocomial infections, including bacteremia, pneumonia, and infections of the skin and urinary tract. A few key clones of A. baumannii-calcoaceticus are currently responsible for the dissemination of these organisms worldwide. Unfortunately, multidrug resistance is a common trait among these clones due to their unrivalled adaptive nature. A. baumannii-calcoaceticus isolates can accumulate resistance traits by a plethora of mechanisms, including horizontal gene transfer, natural transformation, acquisition of mutations, and mobilization of genetic elements that modulate expression of intrinsic and acquired genes.

Pathogen-Targeted Clinical Development to Address Unmet Medical Need: Design, Safety, and Efficacy of the ATTACK Trial.

There is a crucial need for novel antibiotics to stem the tide of antimicrobial resistance, particularly against difficult to treat gram-negative pathogens like Acinetobacter baumannii-calcoaceticus complex (ABC). An innovative approach to addressing antimicrobial resistance may be pathogen-targeted development programs. Sulbactam-durlobactam (SUL-DUR) is a ß-lactam/ß-lactamase inhibitor combination antibiotic that is being developed to specifically target drug-resistant ABC. The development of SUL-DUR culminated with the Acinetobacter Treatment Trial Against Colistin (ATTACK) trial, a global, randomized, active-controlled phase 3 clinical trial that compared SUL-DUR with colistin for treating serious infections due to carbapenem-resistant ABC. SUL-DUR met the primary noninferiority endpoint of 28-day all-cause mortality. Furthermore, SUL-DUR had a favorable safety profile with a statistically significant lower incidence of nephrotoxicity compared with colistin. If approved, SUL-DUR could be an important treatment option for infections caused by ABC, including carbapenem-resistant and multidrug-resistant strains. The development program and the ATTACK trial highlight the potential for pathogen-targeted development programs to address the challenge of antimicrobial resistance.

Durlobactam, a Broad-Spectrum Serine ß-lactamase Inhibitor, Restores Sulbactam Activity Against Acinetobacter Species.

Sulbactam-durlobactam is a pathogen-targeted ß-lactam/ß-lactamase inhibitor combination in late-stage development for the treatment of Acinetobacter infections, including those caused by multidrug-resistant strains. Durlobactam is a member of the diazabicyclooctane class of ß-lactamase inhibitors with broad-spectrum serine ß-lactamase activity. Sulbactam is a first-generation, narrow-spectrum ß-lactamase inhibitor that also has intrinsic antibacterial activity against Acinetobacter spp. due to its ability to inhibit penicillin-binding proteins 1 and 3. The clinical utility of sulbactam for the treatment of contemporary Acinetobacter infections has been eroded over the last decades due to its susceptibility to cleavage by numerous ß-lactamases present in this species. However, when combined with durlobactam, the activity of sulbactam is restored against this problematic pathogen. The following summary describes what is known about the molecular drivers of activity and resistance as well as results from surveillance and in vivo efficacy studies for this novel combination.

Navigating Available Treatment Options for Carbapenem-Resistant Acinetobacter baumannii-calcoaceticus Complex Infections.

Carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRAB) is one of the top-priority pathogens for new antibiotic development. Unlike other antibiotic-resistant threats, none of the available therapies have been shown to consistently reduce mortality or improve patient outcomes in clinical trials. Antibiotic combination therapy is routinely used in clinical practice; however, the preferred combination has not been defined. This narrative review focuses on evidence-based solutions for the treatment of invasive CRAB infections. We dissect the promise and perils of traditional agents used in combination, such as colistin, sulbactam, and the tetracyclines, and offer clinical pearls based on our interpretation of the available data. Next, we investigate the merits of newly developed ß-lactam agents like cefiderocol and sulbactam-durlobactam, which have demonstrated contrasting results in recent randomized clinical trials. The review concludes with the authors' perspective on the evolving treatment landscape for CRAB infections, which is complicated by limited clinical data, imperfect treatment options, and a need for future clinical trials. We propose that effective treatment for CRAB infections requires a personalized approach that incorporates host factors, the site of infection, pharmacokinetic-pharmacodynamic principles, local molecular epidemiology of CRAB isolates, and careful interpretation of antibiotic susceptibility testing results. In most clinical scenarios, a dose-optimized, sulbactam-based regimen is recommended with the addition of at least one other in vitro active agent. Should sulbactam-durlobactam receive regulatory approval, recommendations will need to be re-evaluated with the most recent evidence.

Molecular epidemiology of Acinetobacter baumannii complex causing invasive infections in Korean children during 2001-2020.

BACKGROUND: Acinetobacter baumannii (AB) has emerged as one of the most problematic pathogens affecting critically ill patients. This study aimed to investigate the longitudinal epidemiology of AB causing invasive diseases in children. METHODS: Acinetobacter spp. cultured from sterile body fluids and identified as Acinetobacter calcoaceticus-baumannii (ACB) complexes by automated systems from children aged below 19 years old were prospectively collected during 2001-2020. The discriminative partial sequence of rpoB gene was sequenced to identify the species, and sequence types (STs) were determined. Temporal changes in antimicrobial susceptibilities and STs were analyzed. RESULTS: In total, 108 non-duplicate ACB isolates were obtained from patients with invasive infections. The median age was 1.4 (interquartile range, 0.1-7.9) years, and 60.2% (n = 65) were male. Acinetobacter baumannii comprised 55.6% (n = 60) of the isolates, and the 30-day mortality was higher in patients with isolated AB than in those with non-baumannii Acinetobacter spp. (46.7% vs. 8.3%, P < 0.001). After 2010, complete genotype replacement was observed from non-CC92 genotypes to only CC92 genotypes. Carbapenem resistance rates were highest in AB CC92 (94.2%), followed by AB non-CC92 (12.5%) and non-baumannii Acinetobacter spp. (2.1%). During 2014-2017, which included clustered cases of invasive ST395, colistin resistance increased to 62.5% (n = 10/16), showing a mortality rate of 88% during this period. CONCLUSION: Complete genotype replacement of non-CC92 with CC92 genotypes was observed. AB CC92 was extensively drug-resistant, and pandrug resistance was observed depending on the ST, warranting careful monitoring.

Evaluating the efficiency of a nomogram based on the data of neurosurgical intensive care unit patients to predict pulmonary infection of multidrug-resistant Acinetobacter baumannii.

Background: Pulmonary infection caused by multidrug-resistant Acinetobacter baumannii (MDR-AB) is a common and serious complication after brain injury. There are no definitive methods for its prediction and it is usually accompanied by a poor prognosis. This study aimed to construct and evaluate a nomogram based on patient data from the neurosurgical intensive care unit (NSICU) to predict the probability of MDR-AB pulmonary infection. Methods: In this study, we retrospectively collected patient clinical profiles, early laboratory test results, and doctors' prescriptions (66 variables). Univariate and backward stepwise regression analyses were used to screen the variables to identify predictors, and a nomogram was built in the primary cohort based on the results of a logistic regression model. Discriminatory validity, calibration validity, and clinical utility were evaluated using validation cohort 1 based on receiver operating characteristic curves, calibration curves, and decision curve analysis (DCA). For external validation based on predictors, we prospectively collected information from patients as validation cohort 2. Results: Among 2115 patients admitted to the NSICU between December 1, 2019, and December 31, 2021, 217 were eligible for the study, including 102 patients with MDR-AB infections (102 cases) and 115 patients with other bacterial infections (115 cases). We randomly categorized the patients into the primary cohort (70%, N=152) and validation cohort 1 (30%, N=65). Validation cohort 2 consisted of 24 patients admitted to the NSICU between January 1, 2022, and March 31, 2022, whose clinical information was prospectively collected according to predictors. The nomogram, consisting of only six predictors (age, NSICU stay, Glasgow Coma Scale, meropenem, neutrophil to lymphocyte ratio, platelet to lymphocyte ratio), had significantly high sensitivity and specificity (primary cohort AUC=0.913, validation cohort 1 AUC=0.830, validation cohort 2 AUC=0.889) for early identification of infection and had great calibration (validation cohort 1,2 P=0.3801, 0.6274). DCA confirmed that the nomogram is clinically useful. Conclusion: Our nomogram could help clinicians make early predictions regarding the onset of pulmonary infection caused by MDR-AB and implement targeted interventions.

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection. METHODS: One hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA-51-like and ampC genes, and RAPD-PCR method. RESULTS: All isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA-51-like was detected in all isolates, and blaOXA-23-like was identified in 92.5% of isolates. However, blaOXA-143-like and blaOXA-58-like genes were not detected among isolates. Four distinct blaOXA-51-like alleles were determined as follows: blaOXA-317 (67.0%), blaOXA-90 (9.4%), blaOXA-69 (17.0%), and blaOXA-64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC-88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons. CONCLUSION: The present study demonstrated that there was a high prevalence of blaOXA-23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA-23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

Antibacterial activity of vB_AbaM_PhT2 phage hydrophobic amino acid fusion endolysin, combined with colistin against Acinetobacter baumannii.

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Comparison of mNGS and conventional culture in non-organ transplant critically ill patients supported by ECMO: a single-center study.

Objectives: Infection is one of the important causes of death in intensive care unit (ICU) patients. At present, there are few articles focused on the detailed analysis of pathogenic microorganisms detected in different therapy periods of critically ill patients supported by extracorporeal membrane oxygenation (ECMO). Methods: From October 2020 to October 2022, ECMO-assisted patients who underwent multiple times of both metagenomic next-generation sequencing (mNGS) test and conventional culture were enrolled continuously in the First Affiliated Hospital of Zhengzhou University. The baseline data, laboratory test results, and pathogenic microorganisms detected by mNGS and traditional culture in different time periods were recorded and analyzed. Results: In the present study, 62 patients were included finally. According to whether the patients survived at discharge, they were divided into the survivor group (n = 24) and the non-survivor group (n = 38). Then, according to the different types of ECMO support, they were divided into the veno-venous ECMO (VV ECMO) group (n = 43) and the veno-arterial ECMO (VA ECMO) group (n = 19). The summit period of specimens of traditional culture and mNGS detection of ECMO patients was 7 days after admission, and the largest number of specimens of surviving patients appeared after ECMO withdrawal. The total number of traditional culture specimens was 1,249, the positive rate was 30.4% (380/1,249), and the positive rate of mNGS was 79.6% (82/103). A total of 28 kinds of pathogenic microorganisms were cultured from conventional culture, and 58 kinds of pathogenic microorganisms were detected by mNGS, including Mycobacterium, Rickettsia, and Chlamydia psittaci. In conventional culture, the most frequent Gram-negative bacteria, Gram-positive bacteria, and fungi were Klebsiella pneumoniae, Corynebacterium striatum, and Candida glabrata, and those with the highest frequency of occurrence in mNGS detection were Acinetobacter baumannii, Enterococcus faecium, and Aspergillus flavus. Conclusions: Throughout the whole treatment process, different kinds of suspicious biological specimens of high-infection-risk ICU patients supported by ECMO should undergo both mNGS detection and traditional culture early and repeatedly.

Antimicrobial Resistance in Romania: Updates on Gram-Negative ESCAPE Pathogens in the Clinical, Veterinary, and Aquatic Sectors.

Multidrug-resistant Gram-negative bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, and members of the Enterobacterales order are a challenging multi-sectorial and global threat, being listed by the WHO in the priority list of pathogens requiring the urgent discovery and development of therapeutic strategies. We present here an overview of the antibiotic resistance profiles and epidemiology of Gram-negative pathogens listed in the ESCAPE group circulating in Romania. The review starts with a discussion of the mechanisms and clinical significance of Gram-negative bacteria, the most frequent genetic determinants of resistance, and then summarizes and discusses the epidemiological studies reported for A. baumannii, P. aeruginosa, and Enterobacterales-resistant strains circulating in Romania, both in hospital and veterinary settings and mirrored in the aquatic environment. The Romanian landscape of Gram-negative pathogens included in the ESCAPE list reveals that all significant, clinically relevant, globally spread antibiotic resistance genes and carrying platforms are well established in different geographical areas of Romania and have already been disseminated beyond clinical settings.

Peptaibol Analogs Show Potent Antibacterial Activity against Multidrug Resistant Opportunistic Pathogens.

New classes of antibacterial drugs are urgently needed to address the global issue of antibiotic resistance. In this context, peptaibols are promising membrane-active peptides since they are not involved in innate immunity and their antimicrobial activity does not involve specific cellular targets, therefore reducing the chance of bacterial resistance development. Trichogin GA IV is a nonhemolytic, natural, short-length peptaibol active against Gram-positive bacteria and resistant to proteolysis. In this work, we report on the antibacterial activity of cationic trichogin analogs. Several peptides appear non-hemolytic and strongly active against many clinically relevant bacterial species, including antibiotic-resistant clinical isolates, such as Staphylococcus aureus, Acinetobacter baumannii, and extensively drug-resistant Pseudomonas aeruginosa, against which there are only a limited number of antibiotics under development. Our results further highlight how the modification of natural peptides is a valuable strategy for obtaining improved antibacterial agents with potential therapeutic applications.

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains.

BACKGROUND: Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools. RESULTS: Here, we have constructed a series of all-synthetic allelic exchange vectors - pALFI1, pALFI2 and pALFI3 - with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. CONCLUSIONS: We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.

The evolutionary tale of eight novel plasmids in a colistin-resistant environmental Acinetobacter baumannii isolate.

Acinetobacter baumannii is an important opportunistic pathogen known for its high levels of resistance to many antibiotics, particularly those considered last resorts such as colistin and carbapenems. Plasmids of this organism are increasingly associated with the spread of clinically important antibiotic resistance genes. Although A. baumannii is a ubiquitous organism, to date, most of the focus has been on studying strains recovered from clinical samples ignoring those isolated in the environment (soil, water, food, etc.). Here, we analysed the genetic structures of eight novel plasmids carried by an environmental colistin-resistant A. baumannii (strain E-072658) recovered in a recycled fibre pulp in a paper mill in Finland. It was shown that E-072658 carries a new variant of the mcr-4 colistin resistance gene (mcr-4.7) in a novel Tn3-family transposon (called Tn6926) carried by a novel plasmid p8E072658. E-072658 is also resistant to sulphonamide compounds; consistent with this, the sul2 sulphonamide resistance gene was found in a pdif module. E-072658 also carries six additional plasmids with no antibiotic resistance genes, but they contained several pdif modules shared with plasmids carried by clinical strains. Detailed analysis of the genetic structure of all eight plasmids carried by E-072658 showed a complex evolutionary history revealing genetic exchange events within the genus Acinetobacter beyond the clinical or environmental origin of the strains. This work provides evidence that environmental strains might act as a source for some of the clinically significant antibiotic resistance genes.

Structure of YdjH from Acinetobacter baumannii revealed an active site of YdjH family sugar kinase.

Bacterial sugar kinase is a central enzyme for proper sugar degradation in bacteria, essential for survival and growth. Therefore, this enzyme family is a primary target for antibacterial drug development, with YdjH most preferring to phosphorylate higher-order monosaccharides with a carboxylate terminus. Sugar kinases express diverse specificity and functions, making specificity determination of this family a prominent issue. This study examines the YdjH crystal structure from Acinetobacter baumannii (abYdjH), which has an exceptionally high antibiotic resistance and is considered a superbug. Our structural and biochemical study revealed that abYdjH has a widely open lid domain and is a solution dimer. In addition, the putative active site of abYdjH was determined based on structural analysis, sequence comparison, and in silico docking. Finally, we proposed the active site-forming residues that determine various sugar specificities from abYdjH. This study contributes towards a deeper understanding of the phosphorylation process and bacterial sugar metabolism of YdjH family to design the next-generation antibiotics for targeting A. baumannii.

Characterisation of pellicle-forming ability in clinical carbapenem-resistant Acinetobacter baumannii.

Background: Acinetobacter baumannii was reported to have resistance towards carbapenems and the ability to form an air-liquid biofilm (pellicle) which contributes to their virulence. The GacSA two-component system has been previously shown to play a role in pellicle formation. Therefore, this study aims to detect the presence of gacA and gacS genes in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered from patients in intensive care units and to investigate their pellicle forming ability. Methods: The gacS and gacA genes were screened in 96 clinical CRAB isolates using PCR assay. Pellicle formation assay was performed in Mueller Hinton medium and Luria Bertani medium using borosilicate glass tubes and polypropylene plastic tubes. The biomass of the pellicle was quantitated using the crystal violet staining assay. The selected isolates were further assessed for their motility using semi-solid agar and monitored in real-time using real-time cell analyser (RTCA). Results: All 96 clinical CRAB isolates carried the gacS and gacA genes, however, only four isolates (AB21, AB34, AB69 and AB97) displayed the ability of pellicle-formation phenotypically. These four pellicle-forming isolates produced robust pellicles in Mueller Hinton medium with better performance in borosilicate glass tubes in which biomass with OD570 ranging from 1.984 ± 0.383 to 2.272 ± 0.376 was recorded. The decrease in cell index starting from 13 hours obtained from the impedance-based RTCA showed that pellicle-forming isolates had entered the growth stage of pellicle development. Conclusion: These four pellicle-forming clinical CRAB isolates could be potentially more virulent, therefore further investigation is warranted to provide insights into their pathogenic mechanisms.

Targeting multi-drug-resistant Acinetobacter baumannii: a structure-based approach to identify the promising lead candidates against glutamate racemase.

CONTEXT: Acinetobacter baumannii, one of the critical ESKAPE pathogens, is a highly resilient, multi-drug-resistant, Gramnegative, rod-shaped, highly pathogenic bacteria. It is responsible for almost 1-2% of all hospital-borne infections in immunocompromised patients and causes community outbreaks. Because of its resilience and MDR characteristics, looking for new strategies to check the infections related to this pathogen becomes paramount. The enzymes involved in the peptidoglycan biosynthetic pathway are attractive and the most promising drug targets. They contribute to the formation of the bacterial envelope and help to maintain the rigidity and integrity of the cell. The MurI (glutamate racemase) is one of the crucial enzymes that aid in the formation of the pentapeptide responsible for the interlinkage of peptidoglycan chains. It converts L-glutamate to D-glutamate, which is required to synthesise the pentapeptide chain. METHODS: In this study, the MurI protein of A. baumannii (strain AYE) was modelled and subjected to high-throughput virtual screening against the enamine-HTSC library, taking UDP-MurNAc-Ala binding site as the targeted site. Four ligand molecules, Z1156941329 (N-(1-methyl-2-oxo-3,4-dihydroquinolin-6-yl)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxamide), Z1726360919 (1-[2-[3-(benzimidazol-1-ylmethyl)piperidin-1-yl]-2-oxo-1-phenylethyl]piperidin-2-one), Z1920314754 (N-[[3-(3-methylphenyl)phenyl]methyl]-8-oxo-2,7-diazaspiro[4.4]nonane-2-carboxamide) and Z3240755352 (4R)-4-(2,5-difluorophenyl)-1-(4-fluorophenyl)-1,3a,4,5,7,7a-hexahydro-6H-pyrazolo[3,4-b]pyridin-6-one), were identified to be the lead candidates based on Lipinski's rule of five, toxicity, ADME properties, estimated binding affinity and intermolecular interactions. The complexes of these ligands with the protein molecule were then subjected to MD simulations to scrutinise their dynamic behaviour, structural stability and effects on protein dynamics. The molecular mechanics/Poisson-Boltzmann surface area-based binding free energy analysis was also performed to compute the binding free energy of protein-ligand complexes, which offered the following values -23.32 ± 3.04 kcal/mol, -20.67 ± 2.91kcal/mol, -8.93 ± 2.90 kcal/mol and -26.73 ± 2.95 kcal/mol for MurI-Z1726360919, MurI-Z1156941329, MurI-Z3240755352 and MurI-Z3240755354 complexes respectively. Together, the results from various computational analyses utilised in this study proposed that Z1726360919, Z1920314754 and Z3240755352 could act as potential lead molecules to suppress the function of MurI protein from Acinetobacter baumannii.