Microdilution Minimum Inhibitory Concentration Research Articles

Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a β-lactam/β-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane β-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine β-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of β-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

Genetic Variability and Antimicrobial Susceptibility Profile of Mycoplasma gallisepticum and Antimicrobial Susceptibility Profile of Mycoplasma synoviae Isolated from Various Bird Species in Peninsular Malaysia

Mycoplasma gallisepticum (MG) and M. synoviae (MS) can infect many bird species. Susceptible species of birds are responsible for spillover infections from poultry to wild birds. In Malaysia, previous studies reported the detection of MG in free-flying birds, but there is a lack of information on the characterization and antimicrobial susceptibility profile of these isolates. Therefore, this study aims to molecular characterize and assess the minimum inhibitory concentration (MIC) of MG and MS isolated from different bird species, including aviary, free-flying, and pet birds. Altogether 54 choanal slit swab samples were collected. All the swab samples were subjected to isolation and polymerase chain reaction (PCR) detection. Positive samples were subjected to molecular characterization and MIC assay. Molecular characterization was done using targeted gene sequencing, and microdilution MIC assay was employed to determine the antimicrobial susceptibility profile of the isolates. Using both culture and PCR techniques, 5.6% (3/54) of the samples were positive for MG, while MS was detected in 29.6% (16/54) and 40.7% (22/54) of samples using culture and PCR, respectively. The partial sequence of the mgc2 gene showed a distinct pattern of this gene in one of the MG isolates. Phylogenetic analysis of this isolate indicated that this isolate is a progeny of a Thailand poultry strain. Microdilution MIC assay indicated 100% resistance to erythromycin and chlortetracycline. The findings of this study indicate the possibility of avian interspecies transmission of MG and MS in Malaysia and the need for antimicrobial treatment dosage optimization.

Investigation of Delafloxacin Resistance in Multidrug-Resistant Escherichia coli Strains and the Detection of E. coli ST43 International High-Risk Clone.

Delafloxacin is a novel fluoroquinolone agent that is approved for clinical application. In this study, we analyzed the antibacterial efficacy of delafloxacin in a collection of 47 Escherichia coli strains. Antimicrobial susceptibility testing was performed by the broth microdilution method and minimum inhibitory concentration (MIC) values were determined for delafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem. Two multidrug-resistant E. coli strains, which exhibited delafloxacin and ciprofloxacin resistance as well as extended-spectrum beta-lactamase (ESBL) phenotype, were selected for whole-genome sequencing (WGS). In our study, delafloxacin and ciprofloxacin resistance rates were 47% (22/47) and 51% (24/47), respectively. In the strain collection, 46 E. coli were associated with ESBL production. The MIC50 value for delafloxacin was 0.125 mg/L, while all other fluoroquinolones had an MIC50 value of 0.25 mg/L in our collection. Delafloxacin susceptibility was detected in 20 ESBL positive and ciprofloxacin resistant E. coli strains; by contrast, E. coli strains that exhibited a ciprofloxacin MIC value above 1 mg/L were delafloxacin-resistant. WGS analysis on the two selected E. coli strains (920/1 and 951/2) demonstrated that delafloxacin resistance is mediated by multiple chromosomal mutations, namely, five mutations in E. coli 920/1 (gyrA S83L, D87N, parC S80I, E84V, and parE I529L) and four mutations in E. coli 951/2 (gyrA S83L, D87N, parC S80I, and E84V). Both strains carried an ESBL gene, blaCTX-M-1 in E. coli 920/1 and blaCTX-M-15 in E. coli 951/2. Based on multilocus sequence typing, both strains belong to the E. coli sequence type 43 (ST43). In this paper, we report a remarkable high rate (47%) of delafloxacin resistance among multidrug-resistant E. coli as well as the E. coli ST43 international high-risk clone in Hungary.

Relebactam restores susceptibility of resistant Pseudomonas aeruginosa and Enterobacterales and enhances imipenem activity against chromosomal AmpC-producing species: analysis of global SMART 2018–2020

BackgroundCarbapenem-resistant bacteria are an increasing problem in clinical practice; thus, it is important to identify β-lactamase inhibitors (e.g., relebactam) that can restore carbapenem susceptibility. We report analyses of relebactam enhancement of imipenem activity against both imipenem-nonsusceptible (NS) and imipenem-susceptible (S) Pseudomonas aeruginosa and Enterobacterales. Gram-negative bacterial isolates were collected for the ongoing Study for Monitoring Antimicrobial Resistance Trends global surveillance program. Clinical and Laboratory Standards Institute–defined broth microdilution minimum inhibitory concentrations (MIC) were used to determine the imipenem and imipenem/relebactam antibacterial susceptibilities of P. aeruginosa and Enterobacterales isolates.ResultsBetween 2018 and 2020, 36.2% of P. aeruginosa (N = 23,073) and 8.2% of Enterobacterales (N = 91,769) isolates were imipenem-NS. Relebactam restored imipenem susceptibility in 64.1% and 49.4% of imipenem-NS P. aeruginosa and Enterobacterales isolates, respectively. Restoration of susceptibility was largely observed among K. pneumoniae carbapenemase-producing Enterobacterales and carbapenemase-negative P. aeruginosa. Relebactam also caused a lowering of imipenem MIC among imipenem-S P. aeruginosa and Enterobacterales isolates from chromosomal Ambler class C β-lactamase (AmpC)–producing species. For both imipenem-NS and imipenem-S P. aeruginosa isolates, relebactam reduced the imipenem MIC mode from 16 μg/mL to 1 μg/mL and from 2 μg/mL to 0.5 μg/mL, respectively, compared with imipenem alone.ConclusionsRelebactam restored imipenem susceptibility among nonsusceptible isolates of P. aeruginosa and Enterobacterales and enhanced imipenem susceptibility among susceptible isolates of P. aeruginosa and isolates from Enterobacterales species that can produce chromosomal AmpC. The reduced imipenem modal MIC values with relebactam may result in a higher probability of target attainment in patients.

P024 Genetic determinants of antifungal drug resistance in Fusarium solani species complex

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMDue to its challenging diagnosis and treatment, fungal keratitis is one of the most serious kinds of corneal infection. The Fusarium solani species complex is responsible for nearly half of all fungal keratitis cases. Fusarium infection is difficult to treat because of the increased antifungal resistance of Fusarium species.ObjectiveTo check the antifungal susceptibility in keratitis-causing isolates of F. solani species complex.To find the genetic determinants of resistance using whole genome sequencing.MethodologyPrior to AFST and according to CLSI, clinical isolates of Fusarium species (n = 5) were cultured on potato dextrose agar for 7 days at room temperature. Fungal spores were harvested using 0.8% NaCl and antifungal drug susceptibility testing of 6 different antifungal drugs were tested using CLSI standards broth microdilution method and minimum inhibitory concentrations were obtained. After 24 and 48 h, broth microdilution plates were manually examined. The wells for growth control were also examined. The minimum inhibitory concentration of anti-fungal drugs was the lowest dose that inhibits growth completely (compared to the growth control well). Minimal effective concentration (MEC) was also determined against antifungal drugs. Whole genome sequencing was done using Illumina Hiseqx10. Bioinformatics analysis was done using different bioinformatics tools and software (FASTQC, SPAdes, OmicsBox, AUGUSTUS, etc.).ResultsIn this study the antifungal drug susceptibility from average MIC value were found as fluconazole (512 μg/ml) > itraconazole (32 μg/ml) > amphotericin B (8 μg/ml) > natamycin (8 μg/ml) > posaconazole (2 μg/ml) > voriconazole (1 μg/ml). Fluconazole had a higher MIC value (512 g/ml) in all isolates, however, voriconazole was shown to be more sensitive, with a lower MIC range (0.25-4 g/ml). Glyoxalase/Bleomycin resistance protein, mfs—multidrug resistance transporter, fusaric acid resistance protein, efflux pump antibiotic resistance protein, and copper resistance protein were found by genome-based analysis.ConclusionAs a conclusion of this study, we observed antifungal drug resistance in Fusarium spp., which is often used to treat keratitis in patients, employing fluconazole, itraconazole, and amphotericin B. Resistance to first-line azoles was as a gene variant which contributed to antifungal drug resistance. This study using the phenotypic and genotypic characterization of drug resistance patterns will help to combat antifungal drug resistance.

MIC Discrepancies between Parenteral and Oral Anti-Staphylococcal Beta-Lactams among MSSA

Introduction: Recent evidence has shown that oral antibiotic therapy is not inferior to IV antibiotic therapy in the treatment of complicated Staphylococcus aureus infections. Therefore, oral antibiotic therapy is now frequently prescribed in clinical practice due to cost benefit, ease of administration, decreased complication rate, and lack of need for IV access. In vitro susceptibility testing for β-lactam oral antibiotics is not routinely performed as the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) recommend using oxacillin and cefoxitin as surrogate markers. Hence, oral antibiotic susceptibilities for cephalexin and dicloxacillin are not reported and implied based on oxacillin and cefoxitin. The objective of the current study was to determine whether susceptibilities among S. aureus isolates are predictable when comparing commonly used IV and oral beta-lactams. Methods: Cefazolin, cephalexin, dicloxacillin, and oxacillin broth microdilution minimum inhibitory concentrations (MICs) were determined for 100 clinical isolates of methicillin-sensitive S. aureus by broth microdilution following CLSI guidelines. Results: Among these isolates, median MICs for cephalexin were eight-fold higher than cefazolin MICs and median MICs for dicloxacillin were four-fold less than oxacillin MICs. Ten percent of more strains studied had a major or very major error in its susceptibility reporting when cephalexin was compared to its surrogate marker oxacillin. Discussions/Conclusions: The variations in MICs observed compounded with the dosing and pharmacokinetic differences of oral versus IV β-lactam suggests that establishing breakpoints for oral β-lactam antibiotics is necessary to ensure adequate therapy is selected for the treatment of complex S. aureus infections.

Silver nanoparticle effect on Salmonella enterica isolated from Northern West Egypt food, poultry, and calves

A total no. of 65 Salmonella enterica isolates recovered from food samples, feces of diarrheic calves, poultry, and hospital patient in large five cities at Northern West Egypt were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. The 65 Salmonella enterica isolates had the invA gene were grouped into 11 Salmonella enterica serovars with dominance of S. Enteritidis and S. Kentucky serovars. Their resistance pattern were characterized by using 18 antibiotics from different classes. Approximately 80% of the isolates were multidrug resistant (MDR). Enterobacterial repetitive intergenic consequences polymerase chain reaction (ERIC-PCR) typing of 7 strains of S. Enteritidis showed 5 clusters with dissimilarity 25%. S. Enteritidis clusters in 2 main groups A and B. Group A have 2 human strain (HE2 and HE3) and one food origin (FE7) with a similarity 99%. Group B divided into B1 (FE2) and B2 (FE3) with a similarity ratio ≥ 93%, while ERIC-PCR analysis of 5 strains of S. Kentucky revealed 4 ERIC types, clustered in 2 main groups A and B with similarity 75%. We studied the effect of silver nanoparticles (Ag-NPs) on 10 antibiotic resistant strains of S. Enteritidis and S. Kentucky. The broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected. Evaluation of the affection using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed different ratios of Ag-NPs and microorganism as well as at different contact time ended finally with morphological alteration of the bacteria. We submitted new method in vivo to explore the activity of nanosilver in chicken.Key points• Importance of ERIC-PCR to determine the relatedness between Salmonella isolates.• Effect of silver nanoparticles to confront the antibacterial resistance.• Studying the effect of silver nanoparticles in vivo on infected chicken with Salmonella.

Synthesis, characterization, DNA interactions and biological activity of new palladium(II) complexes with some derivatives of 2-aminothiazoles

Newly palladium(II) complexes (C1, C2) with derivatives of 2-aminothiazoles (L1 = 2-amino-6-methylbenzothiazole, L2 = 2-amino-6-chlorobenzothiazole), general formula [PdL2Cl2] were synthesized and characterized by elemental microanalyses, IR, NMR spectroscopy and X-ray spectroscopy in case of [Pd(L2)2Cl2]. The kinetic of the substitution reactions of complexes and the nucleophiles, such as guanosine-5′-monophosphate (5’-GMP), tripeptide glutathione (GSH) and amino acid L-methionine (L-Met), were studied by stopped-flow technique. The complex C2 was always more reactive, while the order of the reactivity of the nucleophiles, due to the associative mode of the reaction, was L-Met > GSH > 5’-GMP. In order to determine the type of interactions between palladium(II) complexes and calf thymus DNA (CT-DNA), we used electronic absorption spectroscopy, viscosity measurements, and fluorescence spectroscopic studies, while interactions with bovine serum albumin (BSA) were determined only with fluorescence spectroscopic studies. The observed results confirmed that both complexes bound to DNA by groove binding. The significantly strong interaction with BSA, especially for complex C2, was also observed. In vitro cytotoxic activity was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MB-468, HCT116 and mesenchymal stem cells (mMSC). C1 complex showed higher cytotoxic activity against CT26 cell line. Flow cytometry analysis showed that C1 stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule and decelerated proliferation by decreasing Cyclin-D and increasing expression of P21. In vitro antimicrobial activity for ligands and corresponding palladium(II) complexes was investigated by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. Tested compounds exhibited selective and moderate activity.

The Rhodamine B-encrypted Vipericidin Peptide, RhoB-Ctn[1-9], Displays In vitro Antimicrobial Activity Against Opportunistic Bacteria and Yeasts

Crotalicidin (Ctn), a snake venom cathelicidin-related antimicrobial peptide, is a 34-residue-long linear lysine-rich vipericidin obtained from the South American rattlesnake, Crotalus durissus terrificus. Ctn contains tandem repeats of nine amino acid residues (1KRFKKFFKK9 and 16KRLKKIFKK24; consensus: 1KRhKKhFKK9, h = hydrophobic amino acid) as an integral part of its structure. The aim of this study was to evaluate the antimicrobial activity of the encrypted vipericidin nonapeptide KRFKKFFKK, designated as Ctn[1-9], and its structural analogue, rhodamine- B‒conjugated Ctn[1-9], designated as RhoB-Ctn[1-9]. The susceptibility of representative pathogenic bacteria and yeasts to antimicrobial agents was determined using the broth microdilution minimum inhibitory concentration (MIC) method. Cytotoxicity was estimated using a hemolytic assay. The accumulation of RhoB-Ctn[1-9] in microbial cells was observed by fluorescence microscopy. The antimicrobial synergism of RhoB-Ctn[1-9] with antimicrobials was evaluated using a checkerboard analysis. RhoB-conjugated Ctn[1-9] displayed selective antimicrobial activity against infectious gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and pathogenic species of Candida with low hemolytic effects on human erythrocytes which were not observed with unconjugated Ctn[1-9]. RhoB-Ctn[1-9] could permeate cell membranes and accumulate intracellularly in microbial cells. RhoB-Ctn[1-9] exhibits synergistic effects when used with antibiotics or antifungal agents and reduced the MICs of the peptide and antimicrobials. These findings indicate the potential of crotalicidin-related short peptides as structural motifs for the diversification of biological functionalities. Further, they set the stage to investigate the molecular mechanisms by which chemically modified vipericidin repeats modulate cell fate.

1286. Taniborbactam Inhibits Cefepime-Hydrolyzing Variants of Pseudomonas-derived Cephalosporinase (PDC)

BackgroundPDC is a class C β-lactamase in P. aeruginosa. PDC-88 is a variant characterized by a Thr-Pro amino acid deletion in the R2-loop (Δ289-290; Fig. 1). This deletion reduces susceptibility to cefepime (FEP), ceftazidime (CAZ), and ceftolozane-tazobactam (TOL/TZB), but the mechanism for this “gain of function” is unknown. Taniborbactam (TAN) is a novel cyclic boronate β-lactamase inhibitor (BLI) with activity against all four β-lactamase classes and is currently undergoing a phase 3 clinical trial paired with FEP. Herein, we studied the extended-spectrum AmpC (ESAC) phenotype of PDC-88 and examined the ability of TAN to inhibit this variant. Structure of PDC-1 (PDB ID: 4GZB) with PDC-88 deleted residues in red and substitutions in green. All four amino acid substitutions (T79A, V178L, V329I, and G346A) are common (occurring in 10% or more of PDC variants) and have not been associated with resistance. Image rendered using UCSF Chimera.MethodsBroth microdilution minimum inhibitory concentrations (MIC) were determined in accordance with CLSI. PDC-3 and PDC-88 were purified, and steady-state enzyme kinetics were determined. Quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was performed.ResultsIn isogenic E. coli expressing PDC-3 or PDC-88, FEP MIC increased 8- or 128-fold, respectively, compared to the empty vector. Addition of TAN at 4 μg/ml restored FEP activity with MIC lowered to 0.25 μg/ml (Table 1) for both PDC-3 and PDC-88 bearing strains. PDC-88 demonstrated a 9-fold lower KM, 3.4-fold lower kcat, and 2.6-fold higher kcat/KM for FEP compared to PDC-3 (Table 2A). TAN Ki values were 4- to 10-fold lower than avibactam (AVI) and 40- to 95-fold lower than TZB. The TAN acylation constant (k2/K) was 7- to 12-fold greater than AVI and 133- to 366-fold higher than TZB (Table 2B). Q-TOF-MS revealed faster deacylation of FEP by PDC-88 compared to TOL and CAZ. TOL was acylated and deacylated by PDC-88 but not by PDC-3. CAZ was readily acylated but slowly deacylated by PDC-88 compared to PDC-3 (Fig. 2). Cefepime Minimum Inhibitory Concentration (MIC) for PDC-1 and a series of partial R2-loop deletions with and without taniborbactam, avibactam, and tazobactam. In all variants, taniborbactam and avibactam restored susceptiblity while tazobactam is less effective against PDC-88 and variants. Summary of kinetic constants. (A) Comparison of Michaelis constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) of nitrocefin and cefepime with PDC-3 and PDC-88. (B) Comparison of inhibition constant (Ki) and acylation constant (k2/K) for avibactam, tazobactam, and taniborbactam with PDC-3 and PDC-88. Graphical summary of mass spectrometry results for substrate acyl-enzyme complex capture experiments. FEP, cefepime; CAZ, ceftazidime; TOL, ceftolozane. Primes indicate a modified substrate (loss of R2 group). TOL does not form an acyl-enzyme complex with PDC-3.ConclusionDifferent kinetic constants are responsible for the elevated cephalosporin MICs. We posit that PDC-88 increases FEP MIC by enhanced hydrolysis; TOL MICs by enabling acylation; and CAZ MICs by both trapping and enhanced hydrolysis. TAN inhibits both PDC-3 and PDC-88 with similar kinetic profiles. Notably, TAN appears to be a more efficient inhibitor of PDC than current BLIs targeted for use against P. aeruginosa (lower Ki, higher k2/K values). The combination of TAN and FEP may represent an important treatment option for P. aeruginosa isolates that develop ESAC phenotypes.Disclosures Focco van den Akker, PhD, Venatorx Pharmaceuticals, Inc. (Grant/Research Support) Brittany A. Miller, BS, Venatorx Pharmaceuticals, Inc. (Employee) Tsuyoshi Uehara, PhD, Venatorx Pharmaceuticals, Inc. (Employee) David A. Six, PhD, Venatorx Pharmaceuticals, Inc. (Employee) Krisztina M. Papp-Wallace, Ph.D., Merck & Co., Inc. (Grant/Research Support)Spero Therapeutics, Inc. (Grant/Research Support)Venatorx Pharmaceuticals, Inc. (Grant/Research Support)Wockhardt Ltd. (Other Financial or Material Support, Research Collaborator) Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

1110. In Vivo Pharmacodynamics of Vancomycin Against Staphylococci in Young Infants

BackgroundCoagulase-negative staphylococci are the predominant pathogen causing late onset sepsis in young infants, however, the pharmacodynamic target for vancomycin therapy is unknown. This study aimed to determine the pharmacodynamic target of vancomycin in young infants with staphylococcal infections.MethodsRetrospective data were collected for infants aged 0-90 days with methicillin-resistant Staphylococcus aureus (MRSA or coagulase-negative staphylococci (CoNS) bacteraemia over a 4-year period at the Royal Children’s Hospital Melbourne, Australia. Vancomycin broth microdilution minimum inhibitory concentrations (MIC) were determined. A published pharmacokinetic model was externally validated using the study dataset and a time-to-event pharmacodynamic model developed using non-linear mixed effects modelling, with the event being the first negative blood culture. Simulations were performed to determine the 24-hour trough vancomycin concentration correlating with a 90% probability target attainment (PTA) of the area under the curve in the first 24-hours (AUC0-24) exceeding the identified target.ResultsThirty infants, 28 with CoNS and two with MRSA bacteraemia, who had 165 vancomycin concentrations determined were included. The vancomycin broth microdilution MIC was determined for 24 CoNS and one MRSA isolate, both with a median MIC of 1 mg/L (CoNS range 0.5 to 4). An AUC0-24 ≥3 00 mg/L·h was associated with a 7.8-fold increase in the chance of bacteriological cure for all staphylococci at any time point compared to an AUC0-24 < 300 mg/L·h (hazard ratio 95% CI: 3.21-18.8). The 24-hour trough concentrations associated with a 90% PTA of achieving this target were > 13-16 mg/L and > 8-12 mg/L for 6 and 12-hourly dosing, respectively. ConclusionOur study found that an AUC0-24 ≥ 300 mg/L·h was associated with a 7.8-fold increase in bacteriological cure in young infants with staphylococcal bloodstream infections. Disclosures All Authors: No reported disclosures

1063. ARGONAUT-V: Susceptibility of Multidrug-Resistant (MDR) Pseudomonas aeruginosa to Cefepime-Taniborbactam

1063. ARGONAUT-V: Susceptibility of Multidrug-Resistant (MDR) <i>Pseudomonas aeruginosa</i> to Cefepime-Taniborbactam

133. ARGONAUT-III: Susceptibility of Carbapenem-resistant Klebsiellae to Cefepime-Taniborbactam

133. ARGONAUT-III: Susceptibility of Carbapenem-resistant <i>Klebsiellae</i> to Cefepime-Taniborbactam

In Vitro Activity of 22 Antibiotics against Achromobacter Isolates from People with Cystic Fibrosis. Are There New Therapeutic Options?

Bacteria belonging to the genus Achromobacter are increasingly isolated from respiratory samples of people with cystic fibrosis (PWCF). The management of this multidrug-resistant genus is challenging and characterised by a lack of international recommendations, therapeutic guidelines and data concerning antibiotic susceptibility, especially concerning the newer antibiotics. The objective of this study was to describe the antibiotic susceptibility of Achromobacter isolates from PWCF, including susceptibility to new antibiotics. The minimum inhibitory concentrations (MICs) of 22 antibiotics were determined for a panel of 23 Achromobacter isolates from 19 respiratory samples of PWCF. Two microdilution MIC plates were used: EUMDROXF® plate (Sensititre) and Micronaut-S Pseudomonas MIC® plate (Merlin) and completed by a third method if necessary (E-test® or UMIC®). Among usual antimicrobial agents, the most active was imipenem (70% susceptibility). Trimethoprim-sulfamethoxazole, piperacillin and tigecycline (65%, 56% and 52% susceptibility, respectively) were still useful for the treatment of Achromobacter infections. Among new therapeutic options, β-lactams combined with a β-lactamase-inhibitor did not bring benefits compared to β-lactam alone. On the other hand, cefiderocol appeared as a promising therapeutic alternative for managing Achromobacter infections in PWCF. This study provides the first results on the susceptibility of clinical Achromobacter isolates concerning new antibiotics. More microbiological and clinical data are required to establish the optimal treatment of Achromobacter infections.

Synthesis, characterization, HSA/DNA binding, cytotoxicity study, and antimicrobial activity of new palladium(II) complexes with some esters of (S,S)-propylenediamine-N,N'-di-2-(3-methyl)butanoic acid

In this paper, we presented synthesis, characterization, HSA/DNA binding evaluation, in vitro cytotoxic activity and in vitro antimicrobial activity of three new ligands of general formula. R2-S,S-pddmb (L1-L3) and their corresponding palladium(II) complexes of general formula [PdCl2(R2-S,S-pddmb)] (R = n-propyl, n-butyl, n-pentyl; S,S-pddmb = (S,S)-propylenediamine-N,N'-di-2-(3-methyl)-butanoate). Ligands and complexes were characterized by elemental microanalysis, infrared, 1H and 13C NMR spectroscopy. The interactions of new palladium(II) complexes with human serum albumin (HSA) and calf thymus DNA (CT-DNA) were investigated using UV–Vis absorption and fluorescence spectroscopy. The high values of the binding constant, Kb, and the Stern-Volmer quenching constant, KSV, are the result of good binding of all complexes to HSA and CT-DNA. In vitro cytotoxic activity of these ligands and complexes was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MD-468, HCT116 and mesenchymal stem cells (mMSC). C3 complex showed high cytotoxic activity against MDA-MD-468 cell line. Flow cytometry analysis showed that L3 ligand and the corresponding complex (C3) stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule. L3 ligand and C3 complex slowed down cell proliferation and arrested tumor cell in the G0/G1 phase by decreasing Cyclin-D expression and by increasing expression of P21. In vitro antimicrobial activity of ligands and complexes was also investigated. The testing was performed by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. The testing was conducted against 12 microorganisms. The tested ligands and palladium(II) complexes showed selective and moderate activity.

Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing.

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health.

Grapefruit Seed Extract as a Natural Derived Antibacterial Substance against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria are increasing due to the abuse and misuse of antibiotics, and nosocomial infections by MDR bacteria are also increasing. The aim of this study was to identify new substances that can target MDR bacteria among 12 plant extracts that are known to have antibacterial effects. The experiments were performed by the disk diffusion test and microdilution minimum inhibitory concentration (MIC) test, as described by the Clinical and Laboratory Standards Institute (CLSI). By screening against methicillin-sensitive Staphylococcus aureus (MSSA), grapefruit seed extract (GSE) was selected from 12 plant extracts for subsequent experiments. GSE showed antibacterial effects against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) in the disk diffusion test. Even at the lowest concentration, GSE showed antibacterial activity in the microdilution MIC test. As a result, we can conclude that GSE is a naturally derived antibacterial substance that exhibits a favorable antibacterial effect even at a very low concentration, so it is a good candidate for a natural substance that can be used to prevent or reduce nosocomial infections as coating for materials used in medical contexts or by mixing a small amount with other materials.

Development and Application of a Pragmatic Algorithm to Guide Definitive Carbapenemase Testing to Identify Carbapenemase-Producing Pseudomonas aeruginosa.

A minimum inhibitory concentration (MIC) derived algorithm, predictive of carbapenemase production, was developed using a challenge set (n = 92) of Pseudomonas aeruginosa (PA), including carbapenemase-producing (CP), cephalosporinase and/or efflux/porin mutation, and wild-type isolates. Broth microdilution MICs to clinically relevant anti-pseudomonal agents were utilized. The algorithm was applied to 1209 clinical PA isolates from a US surveillance program. Confirmatory genotypic (Xpert® Carba-R assay) and phenotypic (mCIM/eCIM) testing for carbapenemases was conducted on algorithm-derived isolates. With the algorithm, carbapenem resistance alone resulted in poor specificity to identify CP-PA (54%) within the challenge set of isolates. Inclusion of cefepime, ceftazidime, and piperacillin/tazobactam non-susceptibility resulted in a specificity of 66%. Ceftolozane/tazobactam resistance further improved specificity (89%). Of the 1209 isolates, 116 met criteria (carbapenem-resistant and non-susceptibility to cefepime, ceftazidime, and piperacillin/tazobactam) for confirmatory testing. Carba-R and mCIM/eCIM identified five (all blaVIM-positive) and seven carbapenemase-producing isolates, respectively. This MIC algorithm combined with genotypic/phenotypic carbapenemase testing is a pragmatic and streamlined approach to identify CP-PA.

Antibacterial and Antibiofilm Activities of a Traditional Herbal Formula against Respiratory Infection Causing Bacteria

The plants, Althaea officinalis, Tilia cordata and Psidium guaja have been used traditionally to treat respiratory infection symptoms. Flowers of A. officinalis and leaves of T. cordata and P. guaja have been used to treat cough, sore throat, catarrh, oral and pharyngeal mucosa irritation. Therefore, this study was designed to examine the antibacterial and antibiofilm effects of these plants individually as well as in combination, as a formula against respiratory infections causing pathogens. The tested pathogens were Extended Spectrum Beta-Lactamase producing Escherichia coli (ESBL), Beta-Lactamase producing Escherichia coli (BL), Beta-Lactamase producing Klebsiella pneumoniae (BL), Beta-Lactamase producing Pseudomonas aeruginosa (BL), Enterobacter cloacae, and Beta-Lactamase producing Staphylococcus aureus (BL). The tested plants were extracted using ethanol and then fractionated using different polarity solvents (hexane, ethyl acetate and water). Disc diffusion and microdilution (Minimum Inhibitory Concentration) methods were used to evaluate the antibacterial activity while the antibiofilm activity was tested using crystal violet assay. The results showed that A. officinalis and T. cordata extracts and fractions exhibited weak antibacterial activity having MIC values ranged from 6.25 to 12.5 mg/mL. P. guaja exhibited moderate antibacterial activity with MIC values ranged from 6.25 to 1.56 mg/mL. Combination between these plants extracts and fractions in equal proportion provides stronger antibacterial (with MIC values ranged from 6.25 to 0.8 mg/mL) and antibiofilm activities (MBIC50 was 0.2 mg/mL). Therefore, this study provides a valuable scientific knowledge to support the use of plants in combination rather than individually.

Molecular Identification and Antifungal Susceptibility Profiles of Non-albicans Candida Species Clinical Isolates

Background: There is an increasing incidence of life-threatening systemic mycoses, specifically fulminant infections by the Candida species in hospitalised patients and in those who are immunocompromised. Management of the limited number of antifungal drugs currently available requires the identification of infections containing drug-resistant isolates. Objectives: The aim of this study was to identify the non-albicans Candida species as azole-resistant fungi, isolated from sputum and bronchoalveolar lavage specimens of hospitalised cases. Methods: The subjects included hospital-acquired infection (HAI) cases, with a primary diagnosis using a direct microscopic examination, performed for the detection of probable fungi. The molecular tests of PCR-restriction fragment length polymorphism (RFLP) and real-time PCR were performed to confirm the identity and molecular typing of the Candida isolates. Antifungal susceptibility testing (AFST), by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) minimum inhibitory concentration (MIC) (M27-A2) method, was performed on the hospital-isolated Candida species. Results: During 24 months, from August 2014 to September 2016, a total of 198 samples were obtained from cases with proven HAI. The results of experimental studies on the specimens showed 93 (47%) positive cases for a fungal or bacterial infection, of which 54 (58%) had a fungal infection. It was hypothesised that all of the isolated organisms were causative agents of the HAI. Conclusions: The results showed that the medium CHROMagar™ Candida is an accessible and easy-to-use method for the identification of infection, but not as accurate and reliable as PCR-RFLP and real-time PCR methods. Results also showed decreasing susceptibility to azoles (itraconazole in this study) of the Candida species.