Resistant Gram-negative Pathogens Research Articles

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin

BackgroundThe emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. MethodsThe synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. ResultsGigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 μg/ml to 0.25 μg/ml), Salmonella 15E343 (MIC from 8 μg/ml to 1 μg/ml), K. pneumoniae 19-2-1 (MIC from 32 μg/ml to 2 μg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. ConclusionOur results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Clinical epidemiology and case fatality due to antimicrobial resistance in Germany: a systematic review and meta-analysis, 1 January 2010 to 31 December 2021.

BackgroundAntimicrobial resistance (AMR) is of public health concern worldwide.AimWe aimed to summarise the German AMR situation for clinicians and microbiologists.MethodsWe conducted a systematic review and meta-analysis of 60 published studies and data from the German Antibiotic-Resistance-Surveillance (ARS). Primary outcomes were AMR proportions in bacterial isolates from infected patients in Germany (2016-2021) and the case fatality rates (2010-2021). Random and fixed (common) effect models were used to calculate pooled proportions and pooled case fatality odds ratios, respectively.ResultsThe pooled proportion of meticillin resistance in Staphylococcus aureus infections (MRSA) was 7.9% with a declining trend between 2014 and 2020 (odds ratio (OR) = 0.89; 95% CI: 0.886-0.891; p < 0.0001), while vancomycin resistance in Enterococcus faecium (VRE) bloodstream infections increased (OR = 1.18; (95% CI: 1.16-1.21); p < 0.0001) with a pooled proportion of 34.9%. Case fatality rates for MRSA and VRE were higher than for their susceptible strains (OR = 2.29; 95% CI: 1.91-2.75 and 1.69; 95% CI: 1.22-2.33, respectively). Carbapenem resistance in Gram-negative pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp. and Escherichia coli) was low to moderate (< 9%), but resistance against third-generation cephalosporins and fluoroquinolones was moderate to high (5-25%). Pseudomonas aeruginosa exhibited high resistance against carbapenems (17.0%; 95% CI: 11.9-22.8), third-generation cephalosporins (10.1%; 95% CI: 6.6-14.2) and fluoroquinolones (24.9%; 95% CI: 19.3-30.9). Statistical heterogeneity was high (I2 > 70%) across studies reporting resistance proportions.ConclusionContinuous efforts in AMR surveillance and infection prevention and control as well as antibiotic stewardship are needed to limit the spread of AMR in Germany.

Aminoglycosides for the Treatment of Severe Infection Due to Resistant Gram-Negative Pathogens.

Aminoglycosides are a family of rapidly bactericidal antibiotics that often remain active against resistant Gram-negative bacterial infections. Over the past decade, their use in critically ill patients has been refined; however, due to their renal and cochleovestibular toxicity, their indications in the treatment of sepsis and septic shock have been gradually reduced. This article reviews the spectrum of activity, mode of action, and methods for optimizing the efficacy of aminoglycosides. We discuss the current indications for aminoglycosides, with an emphasis on multidrug-resistant Gram-negative bacteria, such as extended-spectrum β-lactamase-producing Enterobacterales, carbapenemase-producing Enterobacterales, multidrug-resistant Pseudomonas aeruginosa, and carbapenem-resistant Acinetobacter baumannii. Additionally, we review the evidence for the use of nebulized aminoglycosides.

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 β-lactam and a β-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.

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase.

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Gram-negative bacilli carrying mcr gene in Brazil: a pathogen on the rise.

The incidence of infections caused by resistant Gram-negative pathogens has become a critical factor in public health due to the limitation of therapeutic options for the control of infections caused, especially, by Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae), Pseudomonas spp., and Acinetobacter spp. Thus, given the increase in resistant pathogens and the reduction of therapeutic options, polymyxins were reintroduced into the clinic. As the last treatment option, polymyxins were regarded as the therapeutic key, since they were one of the few classes of antimicrobials that had activity against multidrug-resistant Gram-negative bacilli. Nonetheless, over the years, the frequent use of this antimicrobial has led to reports of resistance cases. In 2015, mcr (mobile colistin resistance), a colistin resistance gene, was described in China. Due to its location on carrier plasmids, this gene is characterized by rapid spread through conjugation. It has thus been classified as a rising threat to public health worldwide. In conclusion, based on several reports that show the emergence of mcr in different regional and climatic contexts and species of isolates, this work aims to review the literature on the incidence of the mcr gene in Brazil in different regions, types of samples identified, species of isolates, and type of carrier plasmid.

Synergistic Activity of Colistin in Combination with Clofoctol against Colistin Resistant Gram-Negative Pathogens.

Colistin is a bactericidal antibiotic identified decades ago which is active against a number of Gram-negative pathogens. After early elimination from clinical use due to toxicity issues, colistin has been reintroduced as a last-resort treatment for antibiotic-resistant Gram-negative infections lacking other therapeutic options. Inevitably, colistin resistance has emerged among clinical isolates, making the development of colistin adjuvants extremely beneficial. Clofoctol is a synthetic antibiotic active against Gram-positive bacteria, with low toxicity and high tropism for the airways. Interestingly, clofoctol has been found to have multiple biological activities and has been proposed for the treatment of several obstructive lung diseases, including asthma, lung cancer, and SARS-CoV-2 infection. In this study, the activity of clofoctol as a colistin adjuvant was investigated in Gram-negative lung pathogens that are critical for the high prevalence of multidrug-resistant isolates, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Clofoctol potentiated the bactericidal effect of colistin in all tested strains and reduced colistin MICs below the susceptibility breakpoint in nearly all colistin-resistant strains. Overall, this observation supports the development of inhaled clofoctol-colistin formulations for the treatment of difficult-to-treat airway infections caused by Gram-negative pathogens. IMPORTANCE Colistin is used as a last-resort antibiotic against extensively drug-resistant Gram-negative pathogens. However, colistin resistance is on the rise. Clofoctol is an antibiotic used against Gram-positive bacteria, with low toxicity and high penetration and storage in the airways. Here, a strong synergistic activity of the colistin-clofoctol combination against colistin-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii isolates is reported, supporting the development of clofoctol-colistin formulations for the therapy of difficult-to-treat airways infections caused by these Gram-negative pathogens.

The menace of colistin resistance across globe: Obstacles and opportunities in curbing its spread

Colistin-resistance in bacteria is a big concern for public health, since it is a last resort antibiotic to treat infectious diseases of multidrug resistant and carbapenem resistant Gram-negative pathogens in clinical settings. The emergence of colistin resistance in aquaculture and poultry settings has escalated the risks associated with colistin resistance in environment as well. The staggering number of reports pertaining to the rise of colistin resistance in bacteria from clinical and non-clinical settings is disconcerting. The co-existence of colistin resistant genes with other antibiotic resistant genes introduces new challenges in combatting antimicrobial resistance. Some countries have banned the manufacture, sale and distribution of colistin and its formulations for food producing animals. However, to tackle the issue of antimicrobial resistance, a one health approach initiative, inclusive of human, animal, and environmental health needs to be developed. Herein, we review the recent reports in colistin resistance in bacteria of clinical and non-clinical settings, deliberating on the new findings obtained regarding the development of colistin resistance. This review also discusses the initiatives implemented globally in mitigating colistin resistance, their strength and weakness.

Repeated Emergence of Variant TetR Family Regulator, FarR, and Increased Resistance to Antimicrobial Unsaturated Fatty Acid among Clonal Complex 5 Methicillin-Resistant Staphylococcus aureus.

Resistance-nodulation-division (RND) superfamily efflux pumps promote antibiotic resistance in Gram-negative pathogens, but their role in Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) is undocumented. However, recent in vitro selections for resistance of S. aureus to an antimicrobial fatty acid, linoleic acid, and an antibiotic, rhodomyrtone, identified H121Y and C116R substitution variants, respectively, in a TetR family regulator, FarR, promoting increased expression of the RND pump FarE. Hypothesizing that in vivo selection pressures have also promoted the emergence of FarR variants, we searched available genome data and found that strains with FarRH121Y from human and bovine hosts have emerged sporadically in clonal complexes (CCs) CC1, CC30, CC8, CC22, and CC97, whereas multiple FarR variants have occurred within CC5 hospital-associated (HA)-MRSA. Of these, FarRE160G and FarRE93EE were exclusive to CC5, while FarRC116Y, FarRP165L, and FarRG166D also occurred in nonrelated CCs, primarily from bovine hosts. Within CC5, FarRC116Y and FarRG166D strains were polyphyletic, each exhibiting two emergence events. FarRC116Y and FarRE160G were individually sufficient to confer increased expression of FarE and enhanced resistance to linoleic acid (LA). Isolates with FarRE93EE were most closely related to S. aureus N315 MRSA and exhibited increased resistance independently of FarRE93EE. Accumulation of pseudogenes and additional polymorphisms in FarRE93EE strains contributed to a multiresistance phenotype which included fosfomycin and fusidic acid resistance in addition to increased linoleic acid resistance. These findings underscore the remarkable adaptive capacity of CC5 MRSA, which includes the polyphyletic USA100 lineage of HA-MRSA that is endemic in the Western hemisphere and known for the acquisition of multiple resistance phenotypes.

Detection of Colistin Resistance in Gram Negative Pathogens: A One Year Cross-sectional Study in a Tertiary Care Centre in Northeast India

International Journal of Medicine and Public Health,2022,12,4,175-179.DOI:10.5530/ijmedph.2022.4.32Published:January 2023Type:Original ArticleDetection of Colistin Resistance in Gram Negative Pathogens: A One Year Cross-sectional Study in a Tertiary Care Centre in Northeast IndiaDeepayan Biswas, Wihiwot Valarie Lyngdoh, Sheryl Lanong, Clarissa Jane Lyngdoh, Prithwis Bhattacharyya, and Nari Mary Lyngdoh Deepayan Biswas1, Wihiwot Valarie Lyngdoh2, Sheryl Lanong2, Clarissa Jane Lyngdoh2, Prithwis Bhattacharya3, Nari Mary Lyngdoh3 1Department of Microbiology, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA 2Department of Microbiology, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA. 3Department of Anesthesiology and Critical Care, NEIGRIHMS, Mawdiangdiang, Shillong, Meghalaya, INDIA. Abstract:Background: Indiscriminate use of colistin for gram-negative infections has led to increase incidence of colistin resistance. The problem of nosocomial infections especially caused by multi-drug-resistant gram-negative bacteria (MDR-GNB), particularly Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae is a matter of great concern. This study was conducted to find out the prevalence of colistin resistant gram-negative isolates from patients attending outdoor patient department (OPD), those admitted in wards and Intensive care unit from a Tertiary care centre in North-East India. Materials and Methods: Clinical samples obtained were processed using standard microbiological methods. The gram-negative isolates showing colistin resistance by Kirby- Bauer’s disc diffusion method were included and further subjected for MIC (minimum inhibitory concentration) testing by VITEK-2 system followed by confirmation by Broth microdilution method. Results: Colistin resistance was observed in 26 isolates out of 1040 gram-negative isolates using Broth microdilution method. The MIC values varied from 8 to ≥ 32 μg/ml. Majority of them belong to Pseudomonas species followed by Acinetobacter species and were highly resistant to ß-lactams, aminoglycosides, fluoroquinolones. Conclusion: This study highlights an increasing trend of colistin resistance amongst multidrug resistant (MDR) gram-negative isolates warranting routine screening for colistin resistance to guide appropriate therapy for future use. Keywords:Broth microdilution, Colistin, Disc diffusion, Multidrug resistant organisms, VITEK-2View:PDF (346.91 KB)

1999. Cefpodoxime for Spontaneous Bacterial Peritonitis Prophylaxis: Incidence of Breakthrough Infection

Abstract Background Guidelines recommend use of fluoroquinolones and sulfamethoxazole-trimethoprim (SMX-TMP) for spontaneous bacterial peritonitis (SBP) prophylaxis based on data demonstrating a decrease in one-year incidence of SBP. Our institution frequently utilizes cefpodoxime for its favorable safety profile despite the lack of robust clinical experience. We sought to compare rates of breakthrough SBP with the use of cefpodoxime compared to previously reported incidence rates with the use of SMX-TMP and fluoroquinolones. Methods This is an IRB-approved, retrospective, single-center cohort analysis. The electronic medical record at the Hospital of the University of Pennsylvania was utilized to identify cirrhotic patients newly started on cefpodoxime for SBP prophylaxis from March 1, 2016, to December 31, 2020. The primary endpoint was the incidence of breakthrough infections within 1 year in patients who received cefpodoxime for primary or secondary SBP prophylaxis. Secondary endpoints included 1-year all-cause mortality and any microbiologic data from suspected or confirmed SBP during the study timeframe. Descriptive statistics were utilized to analyze baseline characteristics and collected data points. Results Overall, 90 patients met inclusion criteria within our timeframe. The study population consisted of mostly men (65.6%) with a mean age of 59 years. The majority of patients were taking cefpodoxime for primary prophylaxis (66.7%). A large proportion of the patients also had diabetes (44.4%). Alcohol use (53.3%) was the most common cause of cirrhosis, and the mean MELD score was 20.3. Breakthrough SBP occurred in 4 (4.4%) out of 90 patients within 1 year of cefpodoxime initiation. Of the 19 patients who completed a full year of cefpodoxime, 1 (5.2%) patient experienced breakthrough SBP. Microbiologic data was significant for the growth of Citrobacter spp., Streptococcus spp., Enterococcus spp., and Candida glabrata. One patient experienced culture-negative SBP. All-cause mortality at one year in our study was 41.1%. Conclusion Cefpodoxime is a viable option for primary or secondary prophylaxis of SBP based on comparable rates of breakthrough infection. Cefpodoxime does not frequently appear to encourage colonization and infection with resistant gram-negative pathogens. Disclosures All Authors: No reported disclosures.

1715. In Vitro Antibacterial Activity of Cefiderocol against Difficult-to-treat Resistant (DTR) Gram-negative Pathogens in United States from SENTRY Antimicrobial Surveillance Program in 2020/2021

Abstract Background Difficult-to-treat resistant (DTR) isolates are defined as resistance to all first-line high-efficacy, low-toxicity antibiotics (penicillins, cephalosporins, carbapenems and quinolones), leaving physicians with limited treatment options. Cefiderocol (CFDC) is a siderophore cephalosporin with activity against a wide variety of Gram-negative bacteria, including carbapenem-resistant Enterobacterales and non-fermenters. We evaluated the in vitro activity of cefiderocol against DTR isolates collected in United States as part of SENTRY surveillance program in 2020 and 2021. In vitro susceptibility of cefiderocol and comparator agents to DTR isolates Methods Susceptibility testing was performed by broth microdilution according to CLSI guidelines. All antibiotics were tested in cation-adjusted Mueller-Hinton broth (CAMHB) except for CFDC, for which iron-depleted CAMHB was used. Susceptibility rate (%) was determined according to CLSI breakpoints, and DTR pathogens were defined as being resistant to cefepime, ceftazidime ceftriaxone (only for Enterobacterales), imipenem, meropenem, ciprofloxacin and levofloxacin according to CLSI/FDA breakpoints. Results Among a total of 8,328 Enterobacterales, 2,241 Pseudomonas aeruginosa, and 586 Acinetobacter calcoaceticus-baumannii complex (ACB) clinical isolates from the United States, 50 Enterobacterales (0.6%), 36 P. aeruginosa (1.6%) and 114 ACB (19.5%) isolates showed a DTR phenotype, respectively. CFDC demonstrated its potent in vitro activity against these DTR isolates with MIC90 of ≤4 μg/mL with a susceptibility rate of ≥92% except for DTR ACB based on FDA breakpoint (75.4%). In contrast, MIC90s of β-lactam/β-lactamase inhibitor combination drugs showed lower activity (Table). Conclusion Cefiderocol demonstrated potent in vitro activity against DTR isolates of Enterobacterales, P. aeruginosa and ACB, indicating cefiderocol has high potential for treating infections caused by these difficult-to-treat strains. Disclosures Yoshinori Yamano, PhD, Shionogi: Employee Miki Takemura, n/a, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Christine M. Slover, PharmD, Shionogi: Employee Christopher M. Longshaw, PhD, Shionogi: Employee Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant.

139. Development of cefiderocol resistance in an Enterobacter hormaechei strain following prolonged antibiotic exposure

Abstract Background Cefiderocol (FDC) is a novel antimicrobial agent used for multi-drug resistant Gram-negative pathogens. To date, reports of mutations in β-lactamase and siderophore complex genes have been described and may contribute to FDC resistance. This case describes a New Dehli metallo-β-lactamase (NDM)-producing strain of Enterobacter hormaechei that developed FDC resistance following antibiotic exposure. Methods Serial respiratory and blood cultures were collected from a lung transplant recipient throughout 72 days of hospitalization. Confirmatory susceptibility and combination minimal inhibitory concentration (MIC) testing were performed using broth dilution and E-test assays. Short-read sequencing libraries were prepared using a seqWell plexWell 96 kit, and whole-genome sequencing was performed using the Illumina NovaSeq platform. Reads from the sample genomes were aligned to the chromosome and three plasmid sequences of reference genome ENCL48880. Results Four serial respiratory E. hormaechei isolates and one blood isolate were evaluated. Although initial isolates were susceptible to FDC (MICs 1-2 µg/mL), two respiratory isolates cultured after 41 days of FDC therapy had MICs of 128 µg/mL. The blood isolate remained FDC susceptible despite respiratory resistance. The combination of ceftazidime/avibactam and aztreonam was determined to be active via synergy MIC testing in all isolates, and aztreonam therapeutic drug monitoring confirmed an adequate dosing strategy. Whole-genome sequencing revealed no nonsynonymous single nucleotide variants (SNVs) within the chromosomes but identified a deletion of a large urease island in the resistant isolates. In four of the five isolates, a plasmid (p48880_mcr) was identified and analyzed for possible contributions to FDC resistance. Enterobacter Isolate Assemblies This figure demonstrates genomic assemblies from the five Enterobacter clinical isolates, noting an absence of sequence from ECResp2. Conclusion This case demonstrates development of FDC resistance in E. hormaechei isolates during a 41 day course of FDC therapy. Possible causes of resistance include a large chromosomal deletion and plasmid alleles, demonstrating a potential novel mechanism for FDC resistance. Partnering molecular testing and enhanced antimicrobial stewardship should be encouraged to optimize selection of regimens and durations to prevent resistance to FDC. Disclosures Michael G. Ison, MD MS, GlaxoSmithKlein: Grant/Research Support|Pulmocide: Grant/Research Support|Viracor Eurfins: Advisor/Consultant Nathaniel J. Rhodes, PharmD, MSc, American Academy of Colleges of Pharmacy: Grant/Research Support|Paratek: Grant/Research Support|Third Pole Therapeutics: Advisor/Consultant.

1671. Activity of Imipenem/Relebactam and Comparators Against Gram-Negative MDR and DTR Pathogens from Patients with Respiratory and Bloodstream Infections – SMART United States 2018-2020

Abstract Background Gram-negative pathogens with multidrug resistance (MDR) or difficult-to-treat resistance (DTR) are increasingly common, resulting in limited treatment options. These resistant pathogens are often isolated from the respiratory tract or bloodstream and can result in significant patient morbidity and mortality. Imipenem/relebactam is a combination of imipenem with relebactam, a β-lactamase inhibitor of class A and C β-lactamases. We evaluated the activity of imipenem/relebactam and comparators (imipenem, meropenem, piperacillin/tazobactam, cefepime, ceftazidime, levofloxacin, and amikacin) against gram-negative MDR and DTR isolates that were collected from patients with lower respiratory tract (RTI) or bloodstream infections (BSI) in the United States (US). Methods In 2018-2020, 24 clinical labs participated in the global SMART surveillance program in the US, and each collected up to 100 consecutive aerobic or facultative gram-negative pathogens per year from patients with RTI and 50 from BSI. MICs were determined using CLSI broth microdilution and interpreted with CLSI breakpoints. Results MDR isolates were found among 13.5% of collected P. aeruginosa (n=2018), 11.9% of E. coli (n=1952), 13.1% of K. pneumoniae (n=1080), 24.1% of E. cloacae complex (n=460), 19.7% of K. aerogenes (n=304), and 6.1% of S. marcescens isolates (n=304); DTR isolates were found among 7.1%, 0.1%, 2.4%, 1.5%, 0%, and 0.8%, respectively. Imipenem/relebactam was active against 61% of MDR P. aeruginosa, 43-53 percentage points higher than the studied comparator β-lactams, and against 49% of DTR P. aeruginosa, which were nonsusceptible to all studied commonly used β-lactams and levofloxacin (Table). Imipenem/relebactam was also active against 87-100% of MDR Enterobacterales, including against 97% of MDR K. pneumoniae, 18-96 percentage points higher than the studied comparator β-lactams, and against 85% of DTR K. pneumoniae. Conclusion Based on these in vitro data, imipenem/relebactam represents a promising treatment option in the US for patients with RTI or BSI caused by highly resistant gram-negative pathogens that pose substantial treatment challenges. Disclosures Fakhar Siddiqui, MD, MBA, Merck &amp; Co., Inc.: employee|Merck &amp; Co., Inc.: Stocks/Bonds Karri A Bauer, PharmD, Merck &amp; Co., Inc. Merck Research Laboratories: Stocks/Bonds Charles A. DeRyke, PharmD, Merck &amp; Co., Inc. Merck Research Laboratories: Stocks/Bonds Katherine Young, M.S., Merck &amp; Co., Inc.: Stocks/Bonds.

1672. Antimicrobial Activity of Cefepime-Taniborbactam and Comparators Against Clinical Isolates from ICU and Non-ICU Patients; 2018-2020 Global Surveillance

Abstract Background Taniborbactam is a novel cyclic boronate-based broad-spectrum β-lactamase inhibitor with selective direct inhibitory activity against both serine- and metallo-β-lactamases. Taniborbactam restores the activity of cefepime against many difficult to treat organisms, including cephalosporin- and carbapenem-resistant Enterobacterales (EB) and P. aeruginosa (PA). The activity of cefepime-taniborbactam (FTB) and comparator agents was evaluated against clinical isolates of EB and PA from a 2018-2020 global surveillance study. Methods 16,742 isolates of EB and PA were collected from patients in wards designated ICU and non-ICU in 2018-2020. MICs of FTB and comparators were determined by the CLSI reference method. Resistant (R) phenotypes were based on 2022 CLSI breakpoints. Multidrug resistant (MDR) was defined as resistance to at least one agent from ≥3 drug classes. Provisional susceptible (S) breakpoints of ≤8 µg/mL and ≤16 µg/mL were applied for FTB for comparative purposes only. Results FTB had potent activity against EB, with MIC90s of 0.25 µg/mL for isolates from non-ICU patients and 0.5 µg/mL for isolates from ICU patients (Table). At least 99.2% of all EB from both wards were inhibited by ≤8 µg/mL FTB. FTB maintained activity against R phenotypes of EB in both ward types, with 90.3% to 98.1% of isolates inhibited at ≤8 µg/mL. FTB had potent activity against PA, with MIC90 values of 8 µg/mL and inhibiting ≥94.0% of isolates at ≤8 µg/mL for both ICU and non-ICU isolates. FTB maintained activity against R phenotypes of PA in both ward types, with 71.0% to 83.7% of isolates inhibited at ≤8 µg/mL and 84.7% to 92.5% inhibited at ≤16 µg/mL. %S to comparator agents against the R subsets ranged from 1.6% to 92.6% for EB, and 36.0% to 68.4% for PA. Against the particularly challenging subset of MDR PA from ICU patients, FTB was the most active agent tested as it inhibited 73.5/84.7% of isolates at ≤8/≤16 µg/mL compared to 43.1%S to CZA and 36.0%S to C/T. Conclusion Cefepime-taniborbactam demonstrated potent in vitro activity against EB and PA from ICU and non-ICU wards, including resistant subsets of isolates. This supports the continued development of FTB as a potential new treatment option for infections due to resistant Gram-negative pathogens. Disclosures All Authors: No reported disclosures.

PBP Target Profiling by β-Lactam and β-Lactamase Inhibitors in Intact Pseudomonas aeruginosa: Effects of the Intrinsic and Acquired Resistance Determinants on the Periplasmic Drug Availability.

The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or β-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different β-lactams and 4 β-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent β-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized β-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

In vitro activity of cefiderocol against clinically important carbapenem non-susceptible Gram-negative bacteria from Saudi Arabia

Cefiderocol is a novel catechol-substituted siderophore cephalosporin with broad-spectrum activity against Gram-negative pathogens. However, variation of its activity among carbapenemase producers from various regions and countries has been reported. Here, we checked the in vitro activity against Gram-negative carbapenem non-susceptible bacteria collected in Saudi Arabia. Cefiderocol MICs were determined using the iron-depleted cation-adjusted Mueller-Hinton brothand interpreted according to the Clinical and Laboratory Standards Institute guidelines. Isolates (n=288) included carbapenemase-producing Escherichia coli (n=46), Klebsiella pneumoniae (n=98), Acinetobacter baumannii (n=65), and Pseudomonas aeruginosa (n=79) clinical isolates. Cefiderocol inhibited 73.26% (211/288) of the isolates studied at concentrations of ≤ 4 mg/L. Cefiderocol inhibited all carbapenem-resistant A. baumannii isolates (65/65, 100%) producing OXA-23-like, OXA-24-like, and NDM, and nearly all P. aeruginosa isolates (75/79, 94.94%), including those producing VIM and NDM. In contrast, the carbapenemase-producing isolates from the Enterobacterales group demonstrated significantly higher MICs with only 53.06% (52/98) of K. pneumoniae and 41.3% (19/46) of E. coli isolates exhibiting MICs of ≤4 mg/L. Isolates showing elevated MICs (73/144, 50.69%) included NDM (20/29, 68.97%), NDM/OXA-48-like (34/59, 57.63%), OXA-48-like (18/52, 34.62%), and KPC (1/4, 25%) producers, thus showing no clear association with the production of serine-type or metallo-type carbapenemases. However, high cefiderocol MICs (≥ 32mg/L) were associated with isolates producing NDM, and in particular, among those coproducing the OXA-232-type enzyme. Cefiderocol had excellent activity against multi-drug resistant non-fermenting Gram-negative pathogens. Reasons behind the high cefiderocol MICs in certain Enterobacterales isolates need further investigation.

Investigations on In Vivo Pharmacokinetic/Pharmacodynamic Determinants of Fosfomycin in Murine Thigh and Kidney Infection Models.

Background: Amidst the era of widespread resistance, there has been a renewed interest in older antibiotics such as fosfomycin, owing to its activity against certain resistant Gram-negative pathogens, including multidrug-resistant variants expressing extended spectrum β-lactamases or carbapenemases. The goal of the study was to investigate pharmacokinetic/pharmacodynamic (PK/PD) index and PK/PD targets of fosfomycin in murine thigh and kidney infection models, employing clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Methods: Seven isolates of E. coli (one wild-type and six clinical isolates) and five isolates of K. pneumoniae (one wild-type and four clinical isolates) were utilized for in vivo PK/PD studies. Single-dose plasma PK studies were conducted in infected mice by subcutaneous route. PD index was determined from exposure-response analysis employing 24-hr dose fractionation studies in neutropenic murine thigh infection model, while pharmacodynamic targets (PDTs) were derived from both thigh and kidney infection models. Results: Dose fractionation studies demonstrated that in vivo efficacy of fosfomycin best correlated with AUC/MIC for E. coli (R2 = 0.9227) and K. pneumoniae (R2 = 0.8693). The median AUC/MIC linked to 1 log10 kill effects were 346.2 and 745.2 in thigh infection model and 244.1 and 425.4 in kidney infection model for E. coli and K. pneumoniae, respectively. The mice plasma protein binding of fosfomycin was estimated to be 5.4%. Conclusions: The in vivo efficacy of fosfomycin against Enterobacterales was best described by AUC/MIC. The PDTs derived from this study may help define the coverage potential of fosfomycin at the clinical doses approved.

Drug Efflux Pump Inhibitors: A Promising Approach to Counter Multidrug Resistance in Gram-Negative Pathogens by Targeting AcrB Protein from AcrAB-TolC Multidrug Efflux Pump from Escherichia coli

Simple SummaryMultidrug-resistant bacterial infections, especially that caused by Gram-negative bacteria, have posed serious health issues worldwide. Bacteria have different mechanisms that can confer multidrug resistance to bacteria, among these mechanisms are drug efflux pumps that play the main role in conferring multidrug resistance by recognizing then expelling a wide range of compounds, especially antibiotics, and reducing their concentration to sub-toxic levels. Small molecule inhibitors that target drug efflux pumps especially the AcrAB-TolC multidrug efflux pump, from E. coli, appear as a new promising and attractive approach that could increase the required accumulation of antimicrobials to eliminate bacteria as well as leading to reverse antibiotic resistance and prevent the development of resistance in clinically relevant bacterial pathogens and enhances the activity of antibiotics or prolong their effectiveness.Infections caused by multidrug resistance (MDR) of Gram-negative bacteria have become one of the most severe public health problems worldwide. The main mechanism that confers MDR to bacteria is drug efflux pumps, as they expel a wide range of compounds, especially antibiotics. Among the different types of drug efflux pumps, the resistance nodulation division (RND) superfamily confers MDR to various Gram-negative bacteria species. The AcrAB-TolC multidrug efflux pump, from E. coli, a member of RND, is the best-characterized example and an excellent model for understanding MDR because of an abundance of functional and structural data. Small molecule inhibitors that target the AcrAB-TolC drug efflux pump represent a new solution to reversing MDR in Gram-negative bacteria and restoring the efficacy of various used drugs that are clinically relevant to these pathogens, especially in the high shortage of drugs for multidrug-resistant Gram-negative bacteria. This review will investigate solutions of MDR in Gram-negative bacteria by studying the inhibition of the AcrAB-TolC multidrug efflux pump.

A Lysozyme Murein Hydrolase with Broad-Spectrum Antibacterial Activity from Enterobacter Phage myPSH1140.

ABSTRACTBacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multidrug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens, including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.