MICs Of Meropenem Research Articles

Design, Synthesis, and Biological Evaluation of New 1H-Imidazole-2-Carboxylic Acid Derivatives as Metallo-β-Lactamase Inhibitors

As one of important mechanisms to β-lactam antimicrobial resistance, metallo-β-lactamases (MBLs) have been receiving increasing worldwide attentions. Ambler subclass B1 MBLs are most clinically relevant, because they can hydrolyze almost all β-lactams with the exception of monobactams. However, it is still lacking of clinically useful drugs to combat MBL-medicated resistance. We previously identified 1H-imidazole-2-carboxylic acid as a core metal-binding pharmacophore (MBP) to target multiple B1 MBLs. Herein, we report structural optimization of 1H-imidazole-2-carboxylic acid and substituents. Structure-activity relationship (SAR) analyses revealed that replacement of 1H-imidazole-2-carboxylic acid with other structurally highly similar MBPs excepting thiazole-4-carboxylic acid resulted in decreased MBL inhibition. Further SAR studies identified more potent inhibitors to MBLs, of which 28 manifested IC50 values of 0.018 µM for both VIM-2 and VIM-5. The microbiological tests demonstrated that the most tested compounds showed improved synergistic effects; some compounds at 1 µg/ml were able to reduce meropenem MIC by at least 16-fold, which will be worth further development of new potent inhibitors particularly targeting VIM-type MBLs.

1,2,4-Triazole-3-thione analogues with an arylakyl group at position 4 as metallo-β-lactamase inhibitors

Metallo-β-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable β-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 μM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode.

Combatting Planktonic and Biofilm Populations of Carbapenem-Resistant Acinetobacter baumannii with Polymyxin-Based Combinations.

Carbapenem-resistant Acinetobacter baumannii (CRAB) can cause serious infections that are associated with high mortality rates. During the course of an infection, many CRAB isolates are able to form biofilms, which are recalcitrant to several antibiotics and can be difficult to treat. Polymyxin-based regimens are a first-line treatment option for CRAB infections, but they have not been optimized against both planktonic and biofilm phases of growth. The objective of this study was to identify polymyxin-based combinations that are active against planktonic and biofilm populations of CRAB. Four CRAB isolates (meropenem MICs: 8–256 mg/L) capable of forming biofilms were used in each experiment. The activities of polymyxin B alone and in combination with ampicillin/sulbactam, meropenem, minocycline, and rifampin were assessed using time-kill assays, with the CRAB isolates grown in planktonic and biofilm phases. Viable colony counts were used to detect the bactericidal activity and synergy of the antibiotic combinations. Against the planktonic populations, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 3.78, −0.15, 4.38, and 3.23 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Polymyxin B combined with meropenem, ampicillin/sulbactam, or rifampin was synergistic against 75–100% (3/4 or 4/4) of CRAB isolates. Against biofilms, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 1.86, 1.01, 0.66, and 3.55 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Only the combination of polymyxin B and rifampin retained bactericidal activity or synergy against any of the isolates when grown as biofilms (50% of isolates). The combination of polymyxin B and rifampin may be promising for CRAB infections that have planktonic and biofilm populations present.

In vivo translational assessment of the GES genotype on the killing profile of ceftazidime, ceftazidime/avibactam and meropenem against Pseudomonas aeruginosa.

ObjectivesTo evaluate the in vivo killing profile of human-simulated exposures of ceftazidime, ceftazidime/avibactam and meropenem against GES-harbouring Pseudomonas aeruginosa in the murine thigh infection model.MethodsFive P. aeruginosa isolates [three isogenic (GES-1, GES-5 and GES-15) and two clinical (GES-5 and GES-15)] were evaluated. MICs were determined using broth microdilution. Human-simulated regimens (HSRs) of ceftazidime 2 g IV q8h as a 2 h infusion, ceftazidime/avibactam 2.5 g IV q8h as a 2 h infusion and meropenem 2 g IV q8h as a 3 h infusion were administered. Change in bacterial burden relative to baseline was assessed.ResultsModal MICs ranged from 8 to >64 mg/L for ceftazidime, from 1 to 16 mg/L for ceftazidime/avibactam and from 1 to >64 mg/L for meropenem. In vivo, for the isogenic strains, avibactam augmented ceftazidime activity against the GES-1- and GES-15-harbouring isolates. Both ceftazidime and ceftazidime/avibactam resulted in significant kill against the GES-5 isogenic isolate. The meropenem HSR produced >1 log10 kill against each isogenic isolate (MICs of 1–4 mg/L). Against the GES-5 clinical isolate, ceftazidime and ceftazidime/avibactam resulted in >1 log10 kill compared with bacterial growth with the meropenem HSR. In the clinical isolate harbouring GES-15, the elevated MICs of ceftazidime and ceftazidime/avibactam reduced the effectiveness of both compounds, while the observed reduction in meropenem MIC translated into in vivo efficacy of the HSR regimen, predictive of clinical efficacy.ConclusionsIn GES-harbouring P. aeruginosa, quantitative reductions in bacterial density observed with the translational murine model suggest that the phenotypic profile of ceftazidime, ceftazidime/avibactam and meropenem is predictive of clinical efficacy when using the evaluated dosing regimens.

Comparison of carbapenem minimum inhibitory concentrations of Oxacillin-48-like Klebsiella pneumoniae by Sensititre, Vitek 2, MicroScan, and Etest

ObjectivesThe aim of this laboratory-based study was to compare carbapenem MICs yielded by Sensititre, Vitek 2, MicroScan WalkAway plus and Etest for Oxacillin (OXA)-48-like Klebsiella pneumoniae isolates. MethodsAnalysis was performed for categorical agreement for ertapenem, meropenem, and imipenem, and the proportion of isolates with MICs ≤8μg/mL and the MIC50/MIC90 for meropenem and imipenem, from a convenience sample of 82 deduplicated blood culture OXA-48-like K. pneumoniae isolates. ResultsThe proportion of isolates testing susceptible to ertapenem by Etest (19/82, 23.1%) differed from Sensititre/Vitek (0/82) and MicroScan (2/82, 2.4%) (p < 0.001 for all). For meropenem, the proportion of isolates susceptible by Etest (31/82, 37.8%) differed from Sensititre/Vitek (16/82, 19.5%) (p = 0.015). There was variation in the proportion of isolates that tested imipenem susceptible when comparing Sensititre (9/82, 11%) and Vitek (8/82, 9.8%) to MicroScan (27/82, 32.9%), p = 0.001 and p < 0.001, respectively, Sensititre and Vitek to Etest (45/82, 54.9%), p < 0.001 for both, and MicroScan to Etest, p = 0.007. The proportion of isolates with meropenem MICs ≤8μg/mL with Sensititre and Vitek differed significantly from Etest, 58.5% and 85.4%, respectively, p < 0.001. A 2-fold difference between the Sensititre and Vitek meropenem and imipenem MIC at which ≥50% of isolates were inhibited compared to the MicroScan, and a 4-fold difference compared to Etest, was present. ConclusionsSubstantial variability in carbapenem MICs for OXA-48-like carbapenemase-producing Enterobacterales isolates by the four methods was demonstrated. Performance characteristics verification of MIC methods in use for the predominant carbapenemase-producing Enterobacterales type is required by laboratories to optimize the accuracy of carbapenem reporting.

Molecular and biochemical characterization of novel PAM-like MBL variants, PAM-2 and PAM-3, from clinical isolates of Pseudomonas tohonis.

There is no comprehensive study on PAM-like MBLs. Our aim was to characterize novel B3 MBL variants, PAM-2 and PAM-3, from Pseudomonas tohonis clinical isolates. We evaluated the antimicrobial susceptibility and the MBL gene composition of three novel P. tohonis clinical isolates identified at a Japanese hospital, using the broth microdilution method and WGS, respectively. We characterized the PAM-2 and PAM-3 proteins using recombinant protein expression and biochemical evaluations. Low carbapenem MICs (meropenem MIC = 0.125-1 mg/L) were observed for all three P. tohonis isolates; however, the isolates produced MBLs. We identified blaPAM-2 and blaPAM-3 as potential genes, belonging to a novel subclass of B3 MBLs. Their genomic sequence was similar to that of blaPAM-1 from Pseudomonas alcaligenes. PAM-2 and PAM-3 comprised 287 amino acids and exhibited 90% amino acid identity with PAM-1, 73% identity with POM-1 from Pseudomonas otitidis and 61% identity with L1 from Stenotrophomonas maltophilia. Biochemical evaluations of recombinant PAM-2 and PAM-3 revealed similar kcat/Km ratios and demonstrated catalytic activity against all the tested β-lactams, except for aztreonam. In addition, the kcat/Km ratio for imipenem was 40-fold lower than that for meropenem. P. tohonis harbours a species-specific PAM-family MBL gene. This enzyme has higher hydrolytic activity against meropenem compared with that against imipenem.

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3.

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3.

Dihydroxyphenyl-substituted thiosemicarbazone: A potent scaffold for the development of metallo-β-lactamases inhibitors and antimicrobial

The superbug infection mediated by metallo-β-lactamases (MβLs) has grown into anemergent health threat, and development of MβL inhibitors is an ideal strategy to combat the infection. In this work, twenty-five thiosemicarbazones 1a-e, 2a-e, 3a-e, 4a-d, 5a-d and 6a-b were synthesized and assayed against MβLs ImiS, NDM-1 and L1. The gained molecules specifically inhibited NDM-1 and ImiS, exhibiting an IC50 value in the range of 0.37–21.35 and 0.45–8.76 µM, and 2a was found to be the best inhibitor, with an IC50 of 0.37 and 0.45 µM, respectively, using meropenem (MER) as substrate. Enzyme kinetics and dialysis tests revealed and confirmed by ITC that 2a is a time-and dose-dependent inhibitor of ImiS and NDM-1, it competitively and reversibly inhibited ImiS with a Ki value of 0.29 µM, but irreversibly inhibited NDM-1. Structure-activity relationship disclosed that the substitute dihydroxylbenzene significantly enhanced inhibitory activity of thiosemicarbazones on ImiS and NDM-1. Most importantly, 1a-e, 2a-e and 3a-b alone more strongly sterilized E. coli-ImiS and E. coli-NDM-1 than the MER, displaying a MIC value in the range of 8–128 μg/mL, and 2a was found to be the best reagent with a MIC of 8 and 32 μg/mL. Also, 2a alone strongly sterilized the clinical isolates EC01, EC06-EC08, EC24 and K. pneumonia-KPC-NDM, showing a MIC value in the range of 16–128 μg/mL, and exhibited synergistic inhibition with MER on these bacteria tested, resulting in 8–32-fold reduction in MIC of MER. SEM images shown that the bacteria E. coli-ImiS, E. coli-NDM-1, EC24, K. pneumonia-KPC and K. pneumonia-KPC-NDM treated with 2a (64 μg/mL) suffered from distortion, emerging adhesion between individual cells and crumpled membranes. Mice tests shown that monotherapy of 2a evidently limited growth of EC24 cells, and in combination with MER, it significantly reduced the bacterial load in liver and spleen. Docking studies suggest that the 2,4-dihydroxylbenzene of 2a acts as zinc-binding group with the Zn(II) and the residual amino acids in CphA active center, tightly anchoring the inhibitor at active site. This work offered a promising scaffold for the development of MβLs inhibitors, specifically the antimicrobial for clinically drug-resistant isolates.

Association Between Types of Carbapenemase and Clinical Outcomes of Infection Due to Carbapenem Resistance Enterobacterales.

PurposeCompared with non-carbapenemase producing carbapenem-resistant Enterobacterales (non-CP-CRE), carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) are associated with considerable mortality. However, given that the patients are treated with various therapeutic options, it remains unclear whether differences in types of carbapenemase genes yield different mortality rates. Therefore, this study aims to identify carbapenemase genes and identify whether clinical outcomes differ according to the prevalence of genotype and phenotype of carbapenemase among Enterobacterales clinical isolated.Patients and MethodsA retrospective cohort study was performed to determine whether types of carbapenemase genes have an impact on clinical outcomes. Carbapenem-resistant clinical isolates were collected at a tertiary care university hospital in Songkhla, Thailand, between June 2018 and February 2020. Demographic and microbiological data such as antimicrobial susceptibility, carbapenemase genes, and overall mortality were evaluated.ResultsA total of 121 Enterobacterales clinical isolated were evaluated. The blaNDM-1 gene was detected in 44% of the isolates, followed by blaOXA-48 (28%) and blaNDM-1/OXA-48 (28%). NDM-1- or NDM-1/OXA-48- producing isolates were more likely to require meropenem MICs of ≥16 mg/L, while OXA-48-producing isolates were more likely to require meropenem MICs of <16 mg/L. The patients with NDM-1 or NDM-1/OXA-48 had a higher 14 days mortality rate than those with OXA-48 after treating with carbapenem-containing regimens (P-value 0.001) or colistin-containing regimens (P-value < 0.001).ConclusionOur findings suggest that the mortality for CP-CRE infection in patients with NDM-1 or NDM-1/OXA-48 was higher than the mortality in those with OXA-48, which It seems that the type of carbapenemase gene may affect meropenem MIC levels. Hence, in treatment decisions involving the use of either carbapenem-containing regiment or colistin-containing regiment in patients with CP-CRE infection, especially those in the NDM-1 and NDM-1/OXA-48 groups, the patient symptoms should be closely monitored.

The Emergence of Carbapenem Resistant Enterobacteriaceae Producing GIM-1 and SIM-1 Clinical Isolates in Khartoum-Sudan.

PurposeThe aim of this study was to detect multidrug resistant GIM-1 and SIM-1 producing Enterobacteriaceae clinical isolates from hospitalized patients across three Khartoum State Teaching Hospitals, Sudan.Patients and MethodsFrom May 2018 to October 2019, Enterobacteriaceae clinical isolates from inpatients admitted to different Khartoum state hospitals. Genes for carbapenemase (GIM-1 and SIM-1) were amplified by polymerase chain reaction (PCR). Agar dilution method was used to determine MICs for imipenem and meropenem after antimicrobial susceptibility testing.ResultsFive (1.29%) isolates of Enterobacteriaceae [2 (0.51%) Escherichia coli isolates produce GIM-1, 2 (0.51%) Klebsiella pneumoniae isolates (one [0.25%] of each produce of GIM-1 and of SIM-1), and 1 (0.25%) Enterobacter cloacae isolate produce GIM-1]. Susceptibility profiling of the isolates showed a low-level resistance to imipenem and meropenem MICs (8, 16 and 32 μg/mL). It also had resistance to ampicillin, extended-spectrum cephalosporin’s, aztreonam, and amoxicillin-clavulanate and with the two K. pneumoniae strains showing resistance to colistin.ConclusionWe report the emergence of four GIM-1 producing Enterobacteriaceae strains and one strain of SIM-1 producing K. pneumoniae genes, isolated from hospitalized patients, with a high resistance pattern to antimicrobial agents. Whole-genome sequencing (WGS) is necessary for precise identification of clonal diversity backgrounds of acquired carbapenemase genes in diagnostic laboratories as the number of cases of carbapenem resistant Enterobacteriaceae infection increases annually.

In vivo exposure-response relationship of meropenem against metallo-β-lactamase-harbouring Pseudomonas aeruginosa: an assessment using MICs from conventional and zinc-limited broth.

Previous investigations into metallo-β-lactamase (MBL)-harbouring Enterobacterales suggest that susceptibility testing in zinc-limited media may be more appropriate in predicting β-lactam in vivo activity. There are limited data with MBL-harbouring Pseudomonas aeruginosa. Forty-three MBL-harbouring P. aeruginosa isolates (IMP, n = 11; VIM, n = 12; NDM, n = 10; SPM, n = 10) and two P. aeruginosa control isolates (KPC, n = 1; WT, n = 1) were evaluated. Meropenem activity was evaluated in the murine neutropenic thigh model using humanized exposures. Susceptibility testing was conducted in conventional CAMHB, EDTA-supplemented CAMHB (3-300 mg/L EDTA) and Chelex-treated CAMHB (0-1.0 mg/L re-supplemented zinc), resulting in a range of meropenem MIC values for each isolate. A sigmoidal Emax model was fitted to fT>MIC versus change in log10 cfu/thigh to estimate the goodness of fit (R2). Increasing EDTA concentrations or limiting the amount of zinc in broth resulted in several-fold reductions in MIC among the majority of the MBL-harbouring P. aeruginosa while the MICs for the KPC and WT isolates were unchanged. Bacterial killing in vivo was variable, with the range of killing spanning -3.29 to +4.81 log10 change in cfu/thigh. Addition of 30 mg/L EDTA and Chelex-treated CAMHB (with no zinc supplementation) provided broth conditions for susceptibility testing that best predicted in vivo efficacy (R2 > 0.7). Among MBL-harbouring P. aeruginosa, meropenem in vivo efficacy is best represented by the pharmacodynamic profile generated using MICs determined in EDTA-supplemented or zinc-limited broth. In addition to previous data with Enterobacterales, antibiotic susceptibility testing in media that approximates physiological conditions makes it possible to uncover potential and existing therapeutic agents.

The predominance of Klebsiella pneumoniae carbapenemase (KPC-type) gene among high-level carbapenem-resistant Klebsiella pneumoniae isolates in Baghdad, Iraq.

The serine carbapenemase enzymes (KPC) which produce from bacteria klebsiella pneumoniae today have been emerged as one of the β-lactamase enzymes that is capable to inactivating the last line of carbapenems. The gene encoding the K. pneumonia (blaKPC) belongs to gene carried on plasmid among Enterobacteriaceae family, which has modulation for the infections control so this study is aimed to spot the presence and evaluate blaKPC gene expression by real-time PCR in local isolates of K. pneumonia. Forty-seven of K. pneumonia isolates were isolated from different clinical samples (blood, sputum, urine, wounds and burns) from patients in separate hospitals in Baghdad., Antimicrobial sensitivity test was carried out by vitik-2 system and Kirby- Bauer method. The PCR was employed to detect carbapenemase gene. The results of this study showed that all explored isolates were resistant to Ertapenem, Meropenem and imipenem 47(100%). Phenotypically, all the isolates had carbapenemase which hydrolyzed the carbapenem antibiotics. Furthermore, the isolates showed (100%) resistance to Cefazolin, Ampicillin and Amoxicillin/ Clavulic acid. However, the most effective antibiotic was Levofloxacin (91.5%). The results of conventional PCR technique for the detection of blaKPC gene showed that 38 (80.9%) isolates of carbapenem-resistant K. pneumoniae harboured blaKPC gene (1010bp), while none carried other carbapenemase genes including blaNDM1, blaVIM and blaIMP genes. High levels of carbapenem resistance was clarified by the imipenem and meropenem MICs determination. All 38 isolates were positive in CNPT. Furthermore, the 38 isolates showed over expression of blaKPC gene compared with housekeeping rpo gene in Real-Time PCR. According to these results, the resistant isolates to carbapenem were belong to the present and high level expression of blaKPC gene in our local isolates.

Evaluating Mono- and Combination Therapy of Meropenem and Amikacin against Pseudomonas aeruginosa Bacteremia in the Hollow-Fiber Infection Model.

ABSTRACTDebate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied the extent of bacterial killing by and the emergence of resistance to meropenem and amikacin as monotherapies and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs of 0.125, 0.25, and 64 mg/L) were used, simulating bacteremia with an initial inoculum of ~1 × 105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the extent of bacterial killing was increased with the combination regimen compared with the killing by monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the 7-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 h. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa.IMPORTANCE Current guidelines recommend that aminoglycosides should be used in combination with β-lactam antibiotics as initial empirical therapy for serious infections, and otherwise, patients should receive β-lactam antibiotic monotherapy. Given the challenges associated with studying the clinical effect of different antibiotic strategies on patient outcomes, useful data for subsequent informed clinical testing can be obtained from in vitro models like the hollow-fiber infection model (HFIM). Based on the findings of our HFIM, we propose that the initial use of combination therapy with meropenem and amikacin provides some bacterial killing against carbapenem-resistant P. aeruginosa isolates. For susceptible isolates, combination therapy may only be of benefit in specific patient populations, such as critically ill or immunocompromised patients. Therefore, clinicians may want to consider using the combination therapy for the initial management and ceasing the aminoglycosides once antibiotic susceptibility results have been obtained.

In vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii isolated from the urinary tract

The old antimicrobial nitroxoline is currently repurposed for oral treatment of uncomplicated urinary tract infections (UTIs). To investigate the in vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii (CRAb). From an international collection of previously well-characterized clinical A. baumannii isolates, 34 isolates from urinary tract sources with different carbapenem-resistance mechanisms were selected. Nitroxoline activity was analysed with broth microdilution (BMD), disc diffusion (DD) and within an in vitro biofilm model. MICs of meropenem and imipenem were assessed with BMD. Susceptibility to ciprofloxacin and trimethoprim/sulfamethoxazole was investigated using DD. Escherichia coli ATCC 25922 and A. baumannii NCTC 13304 were used for quality control. All isolates were carbapenem resistant (MIC90 >32 mg/L for meropenem and imipenem) and most isolates were resistant to ciprofloxacin (33/34) and trimethoprim/sulfamethoxazole (31/34). Nitroxoline yielded MIC50/90 values of 2/2 mg/L (MIC range 1-2 mg/L) and inhibition zone diameters ranging from 20 to 26 mm. In contrast, for definite eradication of biofilm-associated CRAb in vitro, higher nitroxoline concentrations (≥16 to ≥128 mg/L) were necessary for all isolates. Nitroxoline showed excellent in vitro activity against a collection of CRAb despite high resistance rates to other antimicrobials for parental and oral therapy of A. baumannii UTI. Currently, nitroxoline is recommended for the treatment of uncomplicated UTI in Germany with a EUCAST breakpoint limited to uncomplicated UTI and E. coli (S ≤16 mg/L). Nitroxoline could be a promising drug for oral treatment of lower UTI caused by CRAb. More data are warranted to correlate these findings with in vivo success rates.

Successful Management of a Septic Shock Caused by Acinetobacter baumanii XDR in a Young Heart Transplant Patient

<h3>Introduction</h3> In recent years, <i>Acinetobacter baumannii</i> (Acb) has emerged as a particularly challenging drug-resistant pathogen, but it has rarely been studied in solid organ transplanted patients. <h3>Case Report</h3> We present the case of a 21-year-old female patient who was admitted to our centre for severe cardiac failure due to post-cardiotomy shock. V-A ECMO and Impella were implanted. On admission, the patient presented a SOFA score of 11, an APACHE II score of 24 and a MODS score of 10, with predicted mortality of 50%. Pre-operative blood cultures resulted positive for Acb XDR, with meropenem MIC of 8 mg/l (R) and colistin MIC of 0.25(S). Specific antibiotic therapy with Colistin (4,5 MU x 2/die) and Meropenem (6g i.c. 12.5 ml/h) was started. As general conditions deteriorated, the patient underwent salvage heart transplant (HT).Before HT, in order to reduce the systemic inflammatory response and prevent the multi-organ dysfunction, a CytoSorb cartridge was mounted in the ECMO circuit for 24 h to remove cytokines from the blood. The following day, PCT, CRP and myoglobin started to decrease whereas WCC remained stable. Seven days after HT, the patient developed septic shock with severe hypotension nonresponsive to vasopressors: endotoxin blood level was high (0.72 at EAA). This time, 2 cycles of Toraymyxin hemo-filtration were applied for 48 h. Clinical conditions started to steadily improve, with the reduction of PCR, PCT, WCC levels and body temperature; vasopressor and inotrope dosages were decreased (Tab.1). After 23 days, the patient was transferred to the ward. On the 45th day, the first negative blood culture was obtained, and 20 days later, antibiotic therapy was interrupted. The patient was discharged 6 months after HT. She is reported fine at 27 months of follow-up. <h3>Summary</h3> In this case report, the combined use of Cytosorb, Toraymixyn, targeted antibiotic therapy based on the antibiogram, and modulation of immunosuppressant therapy proved effective in managing such a complex clinical situation.

Disulfiram enhances meropenem activity against NDM- and IMP-producing carbapenem-resistant Acinetobacter baumannii infections.

To evaluate the in vitro and in vivo efficacy of the FDA-approved drug disulfiram in combination with meropenem against MBL-expressing carbapenem-resistant Acinetobacter baumannii. Chequerboard and antibiotic resistance reversal analysis were performed using 25 clinical isolates producing different MBLs. Three representative strains harbouring NDM, IMP or non-MBL genes were subjected to a time-kill assay to further evaluate this synergistic interaction. Dose-dependent inhibition by disulfiram was assessed to determine IC50 for NDM-1, IMP-7, VIM-2 and KPC-2. Further, to test the efficacy of meropenem monotherapy and meropenem in combination with disulfiram against NDM- and IMP-harbouring A. baumannii, an experimental model of systemic infection and pneumonia was developed using BALB/c female mice. Chequerboard and antibiotic reversal assay displayed a synergistic interaction against MBL-expressing A. baumannii strains with 4- to 32-fold reduction in MICs of meropenem. In time-kill analysis, meropenem and disulfiram exhibited synergy against NDM- and IMP-producing carbapenem-resistant A. baumannii (CRAb) isolates. In vitro dose-dependent inhibition analysis showed that disulfiram inhibits NDM-1 and IMP-7 with IC50 values of 1.5 ± 0.6 and 16.25 ± 1.6 μM, respectively, with slight or no inhibition of VIM-2 (<20%) and KPC-2. The combination performed better in the clearance of bacterial load from the liver and spleen of mice infected with IMP-expressing CRAb. In the pneumonia model, the combination significantly decreased the bacterial burden of NDM producers compared with monotherapy. These results strongly suggest that the combination of disulfiram and meropenem represents an effective treatment option for NDM- and IMP-associated CRAb infections.

Risedronate and Methotrexate Are High-Affinity Inhibitors of New Delhi Metallo-β-Lactamase-1 (NDM-1): A Drug Repurposing Approach.

Bacteria expressing New Delhi metallo-β-lactamase-1 (NDM-1) can hydrolyze β-lactam antibiotics (penicillins, cephalosporins, and carbapenems) and, thus, mediate multidrug resistance. The worldwide dissemination of NDM-1 poses a serious threat to public health, imposing a huge economic burden in the development of new antibiotics. Thus, there is an urgent need for the identification of novel NDM-1 inhibitors from a pool of already-known drug molecules. Here, we screened a library of FDA-approved drugs to identify novel non-β-lactam ring-containing inhibitors of NDM-1 by applying computational as well as in vitro experimental approaches. Different steps of high-throughput virtual screening, molecular docking, molecular dynamics simulation, and enzyme kinetics were performed to identify risedronate and methotrexate as the inhibitors with the most potential. The molecular mechanics/generalized Born surface area (MM/GBSA) and molecular dynamics (MD) simulations showed that both of the compounds (risedronate and methotrexate) formed a stable complex with NDM-1. Furthermore, analyses of the binding pose revealed that risedronate formed two hydrogen bonds and three electrostatic interactions with the catalytic residues of NDM-1. Similarly, methotrexate formed four hydrogen bonds and one electrostatic interaction with NDM-1’s active site residues. The docking scores of risedronate and methotrexate for NDM-1 were –10.543 kcal mol−1 and −10.189 kcal mol−1, respectively. Steady-state enzyme kinetics in the presence of risedronate and methotrexate showed a decreased catalytic efficiency (i.e., kcat/Km) of NDM-1 on various antibiotics, owing to poor catalytic proficiency and affinity. The results were further validated by determining the MICs of imipenem and meropenem in the presence of risedronate and methotrexate. The IC50 values of the identified inhibitors were in the micromolar range. The findings of this study should be helpful in further characterizing the potential of risedronate and methotrexate to treat bacterial infections.

Phenotype-genotype correlations among carbapenem-resistant Enterobacterales recovered from four Egyptian hospitals with the report of SPM carbapenemase

BackgroundCarbapenem-resistant Enterobacterales (CRE), currently listed by the World Health Organization (WHO) as top priority critical pathogens, are a major global menace to human health. In low- and middle-income countries (LMICs) the threat is mounting fueled by selective pressures caused by antibiotic abuse and inadequate diagnostic resources.MethodsThis study phenotypically and genotypically characterized carbapenem resistance among 115 Enterobacterales isolates including 76 Klebsiella (K.) pneumoniae, 19 Escherichia (E.) coli, 14 Shigella (S.) sonnei, 5 Enterobacter (E.) cloacae, and 1 Proteus (P.) mirabilis.ResultsNinety-three isolates (80.9%) were carbapenem-resistant with an alarming 57.5% carbapenem non-susceptibility in isolates collected from the outpatient department. Molecular characterization of the carbapenemases (CPases) encoding genes showed that blaNDM (80.5%) was the most prevalent; it was detected in 62 isolates (54 K. pneumoniae, 6 E. coli and 2 S. sonnei), followed by blaVIM (36.4%) which was observed in 28 isolates (24 K. pneumoniae, 3 E. coli and 1 E. cloacae). Other CPases included blaKPC (28.6%; in 20 K. pneumoniae, 1 E. coli and 1 S. sonnei), blaOXA-48 (26%; in 17 K. pneumoniae, 1 E. coli,1 E. cloacae and 1 P. mirabilis), blaIMP (6.5%; in 5 K. pneumoniae) and blaSPM (1.3%; in K. pneumoniae). Notably more than half of the Enterobacterales isolates (54.5%) co-harboured more than one CPase-encoding gene. Co-existence of blaNDM and blaVIM genes was the most dominant (31.2%), followed by association of blaNDM and blaKPC (24.7%), then blaVIM and blaKPC (13%). Moreover, the effects of different genotypes on meropenem MIC values were assessed, and a statistically significant difference between the genotype (Ambler classes A and B) and the genotype (Ambler classes B and D) was recorded.ConclusionThe current findings may serve for a better understanding of the context of CRE in Egypt, associated drivers and CPases.

Role of AxyABM overexpression in acquired resistance in Achromobacter xylosoxidans.

Acquired antimicrobial resistance among Achromobacter isolates from cystic fibrosis (CF) patients is frequent. Data concerning the mechanisms involved are scarce. The role of the AxyXY-OprZ and AxyEF-OprN Resistance Nodulation Division (RND) efflux systems has been demonstrated, but not that of AxyABM. To explore the role of efflux systems in the acquired multiresistance observed in a one-step mutant selected after ofloxacin exposure. The in vitro resistant mutant NCF-39-Bo2 and its parental strain NCF-39 (MICs of meropenem of 8 and 0.19 mg/L, of ceftazidime of 12 and 3 mg/L, of cefiderocol of 0.094 and 0.032 mg/L and of ciprofloxacin of 8 and 1.5 mg/L, respectively) were investigated by RNA-seq and WGS. Gene inactivation and reverse transcription quantitative PCR (RT-qPCR) were used to explore the role of the efflux systems of interest. RNA-seq showed that the AxyABM efflux system was overproduced (about 40-fold) in the in vitro mutant NCF-39-Bo2 versus its parental strain NCF-39. A substitution in AxyR, the putative regulator of AxyABM, was detected in NCF-39-Bo2. Gene inactivation of axyB (encoding the transporter component) in NCF-39-Bo2 led to a decrease in MICs of ciprofloxacin (5-fold), meropenem (64-fold), ceftazidime (12-fold) and cefiderocol (24-fold). Inactivation of axyB in the clinical isolate AXX-H2 harbouring a phenotype of resistance close to that of NCF-39-Bo2 enhanced the activity of the same molecules, especially meropenem. AxyABM overproduction is involved in acquired resistance of Achromobacter to ciprofloxacin, meropenem and ceftazidime, antibiotics widely used in CF patients, and increases the MIC of the new promising antibiotic cefiderocol.

1285. Identification of KPC Omega Loop Variant R163S Conferring Ceftazidime-Avibactam Resistance

BackgroundWe report on a 56 year-old male with prolonged COVID-19 pneumonia who initially improved with dexamethasone and intubation but quickly decompensated. Clinical and radiologic features were consistent with VAP. Tracheal aspirate cultures grew carbapenem-resistant Enterobacter cloacae; meropenem (MEM) MIC was >8 ug/ml (resistant) while ceftazidime-avibactam (CZA) MIC was 2/4 ug/ml (susceptible). Lateral flow antigen assay detected a KPC enzyme. The patient was treated with CZA with steady improvement in respiratory function over the next two weeks. He then experienced an episode of tachycardia, prompting repeat culture. At this point the patient had been extubated: sputum culture grew KPC+ E. cloacae that now showed CZA-resistance (MIC >8/4 ug/ml) and paradoxical decrease in MEM MIC (4 ug/ml); meropenem-vaborbactam (< 2/8 ug/ml) was susceptible.MethodsThe pre- & post-CZA therapy E. cloacae isolates underwent whole genome sequencing using the Illumina 150bp paired end protocol; sequences were quality trimmed and compared.ResultsA point mutation in the plasmid blaKPC3 gene was identified in the post-CZA therapy isolate, an R163S mutation in the omega loop of the enzyme. ompC and ompF porin genes were analyzed to rule-out decreased influx as a mechanism for CZA-resistance: the pre- and post-CZA isolates had identical porin sequences.ConclusionThis case highlights emerging mutations within KPC carbapenemases that lead to resistance to ‘last-line’ antimicrobials like CZA. The presumptive mechanism is increased KPC active site promiscuity due to increased omega loop flexibility, allowing increased ceftazidime binding and hydrolysis, and decreased avibactam binding and beta lactamase inhibition. Paradoxically, MEM susceptibility improves after such omega loop mutations, likely due to decreased active site binding affinity, a ‘seesaw’ effect between MEM and CZA. While authors have reported MEM MICs falling into the ‘susceptible’ category after an omega loop variant, these bacteria invariably develop secondary mutations leading to MEM treatment failure. Fortunately, given our patient’s improved respiratory status, the post-CZA E. cloacae isolate was felt to reflect colonization and the patient was discharged home without antimicrobial therapy.Disclosures Romney Humphries, PhD D(ABMM), Accelerate Diagnostics (Individual(s) Involved: Self): Consultant, Shareholder; IHMA (Individual(s) Involved: Self): Consultant; Melinta (Individual(s) Involved: Self): Consultant; Momentum (Individual(s) Involved: Self): Grant/Research Support; Pattern (Individual(s) Involved: Self): Consultant; QPex (Individual(s) Involved: Self): Consultant; ThermoFisher (Individual(s) Involved: Self): Consultant; Torus (Individual(s) Involved: Self): Consultant