Mueller-Hinton Broth Research Articles

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.

660. In vitro activity of cefiderocol against difficult-to-treat resistance European Gram-negative bacterial pathogens from the multi-national sentinel surveillance study, SENTRY in 2020 and 2021

Abstract Background Difficult-to-treat resistance (DTR) organisms are defined as non-susceptible to all first-line high-efficacy, low-toxicity antibiotics (penicillins, cephalosporins, carbapenems and fluoroquinolones), leaving physicians with limited treatment options. Cefiderocol is a parenteral siderophore cephalosporin with potent activity against aerobic Gram-negative pathogens, including carbapenem-resistant strains. We evaluated the in vitro activity of cefiderocol and comparators against DTR pathogens collected in Europe by the SENTRY surveillance study in 2020 and 2021. Methods A total of 11434 clinical isolates of Gram-negative bacilli were systematically collected from 16 EU countries, Israel and Turkey in 2020 and 2021. Minimum inhibitory concentrations (MICs) were determined by broth microdilution for a panel of twenty-two antibiotics according to CLSI guidelines. All antibiotics were tested in cation-adjusted Mueller-Hinton broth (CAMHB) except for cefiderocol, for which iron-depleted CAMHB was used. Susceptibility was determined according to CLSI breakpoints, and DTR pathogens were defined as being resistant to cefepime, ceftazidime ceftriaxone, imipenem, meropenem, ciprofloxacin and levofloxacin according to CLSI breakpoints. Results Among 11434 Gram-negative isolates collected in 2020 and 2021, 792 (7.0%) were resistant to all 1st line therapy including cephalosporins, carbapenems and fluoroquinolone and could be defined as DTR. DTR was most frequently observed in Acinetobacter spp. (530/931, 56.9%), Enterobacterales (201/7739, 2.6%) and Pseudomonas aeruginosa (61/2440, 2.5. Based on CLSI breakpoints, cefiderocol was the most active antibiotic tested against DTR-Acinetobacter spp. (MIC90= 2mg/L, 97.4% susceptibility). Ampicillin/sulbactam was active in less than 1% of the DTR-Acinetobacter spp isolates. None of the drugs recommended by the IDSA for the treatment of resistant Gram-negative infections were as potent as cefiderocol (Table 1). Conclusion Cefiderocol was the only treatment option with demonstrated in vitro activity against more than 95% of all the tested DTR Gram-negative pathogens with limited treatment options. Disclosures Anne Laurence Santerre Henriksen, PhD, Shionogi: Contractor|UTILITY therapeutics Ltd: Advisor/Consultant Christopher M. Longshaw, PhD, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Miki Takemura, n/a, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi: Employee.

1734. Susceptibility of Cefiderocol between US Census Regions against Gram-Negative Organisms collected from the SENTRY Surveillance Program: 2020-2021

Abstract Background Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with broad activity against Gram-negative (GN) bacteria, including multidrug-resistant organisms. GN bacteria such as Enterobacterales (ENT), Pseudomonas aeruginosa (PsA), Acinetobacter baumannii complex (ABC), and Stenotrophomonas maltophilia (StM) can be challenging to treat and are often carbapenem-resistant (CR). Regional susceptibility of CFDC and comparators were investigated against US GN isolates collected in 2020-2021 as part of the SENTRY Antimicrobial Surveillance Program. Methods GN pathogens were consecutively collected from 32 US hospitals between 2020 to 2021. Susceptibility testing was performed using the broth microdilution method. CFDC was tested in iron-depleted cation-adjusted Mueller-Hinton broth. FDA or CLSI breakpoints were used where available. Other agents tested included the beta-lactam/beta-lactamase inhibitor (BL/BLI) combinations ceftazidime-avibactam, ceftolozane-tazobactam, imipenem-relebactam, meropenem-vaborbactam, piperacillin-tazobactam, and ampicillin-sulbactam. CR-PsA, or CRAB was defined as meropenem resistant while CRE was defined as imipenem or meropenem resistant by CLSI breakpoints. Results A total of 8328 ENT, 2241 PsA, 586 ABC, and 404 StM were collected. For ENT and PsA, CFDC susceptibility between the 9 US Census regions remained above 98% susceptible by CLSI or FDA breakpoints in all regions (Table 1). Amongst the BL/BLI combinations, the majority had >90% susceptibility across regions except for piperacillin-tazobactam with 79.7% for ENT and 65.9% for PsA in the MidAtlantic by FDA breakpoints. For ABC and StM, CFDC susceptibility was > 88% across all regions. For CR pathogens, 96 CRE, 327 CR-PsA, and 199 CRAB were collected. CFDC had >87.5% susceptibility in all regions for CRE and CR-PSA. For CRAB, 4 regions were > 87.5% by FDA breakpoints and the lowest was 63.6% in 11 isolates in the Mountain region (Table 2). For CRAB, every BL/BLI had susceptibility ≤27.3% in every region. Ampicillin-sulbactam had 27.3% susceptibility in the Mountain region. Table 1 Susceptibility of Cefiderocol against Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii complex and Stenotrophomonas maltiphilia isolates in the cefiderocol program collected from medical centers in the USA Table 2: Susceptibility of cefiderocol against CRE, CR Pseudomonas aeruginosa, and CR Acinetobacter baumannii complex isolates in the cefiderocol program collected from medical centers in the USA Conclusion US GN isolates, including CR pathogens, had high susceptibilities to CFDC across the US census regions. CFDC remains an important treatment option for GN infections in all US census regions. Disclosures Frank H. Kung, PhD, Shionogi Inc: Employee Sean Nguyen, n/a, Shionogi: Employee Christine M. Slover, PharmD, Shionogi: Employee Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Roger Echols, MD, Shionogi: Advisor/Consultant Miki Takemura, n/a, Shionogi: Employee Yoshinori Yamano, PhD, Shionogi: Employee.

339. Feasibility of Heteroresistance Screening on Escherichia coli (EC) to Predict the Presence of Inner Colonies (IC) During Fosfomycin Disk Diffusion (DD) Testing

Abstract Background Growth of IC within the zone of inhibition during fosfomycin DD testing in EC are found, but CLSI and EUCAST interpretation criteria for IC are contradictory. There is a need to determine appropriate methods of interpretation to prevent resistant infections, thus evaluating the susceptibility of IC EC isolates in comparison to parent can help determine optimal methods for DD testing. A heteroresistance screening test has potential to identify isolates with IC. We sought to determine the feasibility of heteroresistance screening to predict the presence of IC in clinical EC isolates. Methods A convenience collection (n=48) of EC parent isolates underwent fosfomycin DD testing to identify those with IC. Broth microdilution (BMD) testing was performed on all parent and IC (n=34) to determine minimal inhibitory concentrations (MICs) to establish inclusion criteria for heteroresistance screening. A disk elution test for heteroresistance screening was performed in duplicate on separate days. In tubes of Mueller-Hinton broth, 6 commercial fosfomycin disks (each 200 μg fosfomycin and 50 μg glucose-6-phosphate) were eluted for 90 minutes. One hundred microliters of each bacterial isolate from an overnight culture was suspended in tubes. A positive test was a turbid tube after 72 hours of incubation. Results The parent isolates (n=48) had an MIC range of 1 to >256 μg/mL with a median of 4 μg/mL. The subset of IC isolates (n=34) had an MIC range between ≤8 and >1024 with a median of 128 μg/mL. Nineteen IC isolates met the inclusion criteria for the heteroresistance screening; 5 isolates did not produce IC (MIC range of 1-32 μg/mL) while 14 isolates produced IC (MIC range of 1-16 μg/mL). Of these 19 isolates, 6 (32%) were heteroresistant using the disk elution test. Of those with IC, 4 (29%) were heteroresistant while 2 (40%) isolates without IC were heteroresistant. Conclusion A heteroresistance screening test did not provide consistent data to predict the presence of IC among this EC collection. A larger isolate set and further studies are needed to understand the feasibility of a heteroresistance screening test and increased resistance in IC resulting from fosfomycin DD testing among EC. Disclosures Elizabeth B. Hirsch, PharmD, FCCP, FIDSA, Melinta: Advisor/Consultant|MeMed: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 μg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 μg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 μg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

In vitro Activity of Cefiderocol and Comparators against Carbapenem-Resistant Gram-Negative Pathogens from France and Belgium.

Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller–Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates.

Analysis of In Vitro Activity of Cefiderocol Against Carbapenem-Resistant Gram-Negative Bacilli by Broth Microdilution and Disk Diffusion Method: A Single-Center Study in Odisha, India

IntroductionCefiderocol (CFDC), a novel semi-synthetic siderophore cephalosporin has been developed to combat the menace of infections caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) including Carbapenem-resistant Enterobacterales (CRE) and Carbapenem-resistant Nonfermenting Gram-negative bacilli (CR-NFGNB).MethodsWe determined the in vitro activity of CFDC against a contemporary collection of 503 CR-GNB isolates by the reference broth microdilution method (BMD) using Iron depleted cation adjusted Mueller-Hinton broth (ID-CAMHB). Performance of CFDC disk diffusion (DD) was evaluated against the reference BMD, as an alternative convenient testing method. Molecular characterization of carbapenemase in CR-GNB was performed by PCR targeting blaNDM-1, blaOXA-48like alleles, blaKPC, blaIMP, and, blaVIM. Minimum inhibitory concentration (MIC) distribution of CFDC in CR-GNB harbouring different carbapenemase enzymes was also analyzed.ResultsIn our study, 81.7% (411/503) of CR-GNB isolates [81.3%, (278/342) CRE and 82.6% (133/161) CR-NFGNB] were susceptible to CFDC (p>0.05). Categorical agreement (CA) of DD ranged from 79.8% to 87.5%, Minor error (mE) ranged from 0 to 14%, Major error (ME) ranged from 0 to 3.5%, and Very Major error (VME) ranged from 0 to 12.5% with variations by species tested. Overall CFDC MIC50 and MIC90 values of CR-GNB isolates without any carbapenemase genes were higher as compared to those with the presence of carbapenemase genes (4 µg/mL and 128 µg/mL versus 2 µg/mL and 16 µg/mL respectively).DiscussionCFDC is not yet available for clinical use in India. Hence, multicentric studies are the need of the hour in India for standardization of CFDC susceptibility using disks and CAMHB from different manufacturers as well as understanding mechanisms of high MIC values.

Culture media, DMSO and efflux affect the antibacterial activity of cisplatin and oxaliplatin.

Cisplatin was originally discovered through its antibacterial action and subsequently has found use as a potent broad-spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. Escherichia coli MG1655 or MG1655 ΔtolC was treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by fourfold (MIC 12·5 mg l-1 ) compared with 0·9% NaCl-solubilized cisplatin (MIC 3·12 mg l-1 ) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller-Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur-containing amino acids, l-cysteine and l-methionine, individually or in combination reduced activity by ≥8-fold (MIC ≥25 mg l-1 ). Oxaliplatin was less active against E.coli (MIC 100 mg l-1 ) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with l-cysteine and l-methionine (MIC ≥400 mg l-1 ). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII.

Evaluation of Colistin Susceptibility Results in Multidrug-Resistant Gram-Negative Bacilli by Colistin Broth Disk Elution Method

Objective: Determining colistin susceptibility according to the reference method is a significant problem for clinical microbiology laboratories. There is a need for a fast, easy-to-apply, and accurate method for laboratories with limited opportunities. In our study, we aimed to evaluate the applicability of the colistin broth disk elution (CBDE) test. Methods: A total of 193 Gram-negative bacteria, which were isolated from various clinical specimens in our laboratory, were included in the study. Colistin susceptibility was determined by broth microdilution as the reference method. The CBDE test was performed as previously described. For each bacterial isolate, 0, ½, 1, 2, and 4 colistin discs (10 µg; BD) were added to 5 tubes containing cation-adjusted Mueller-Hinton broth (MHB). The final colistin concentrations in the tubes were 0 (growth control), 0.5, 1, 2, and 4 µg/ml, respectively. The minimum inhibitory concentration values were determined by visual evaluation 16-20 hours after the bacterial inoculation. Results: In the Enterobacterales group, the categorical agreement was 94%, the major error was 5%, and the very major error was 0%. In the non-fermentative bacteria group, the categorical agreement was found to be 94%, the major error 1%, and the very major error 33%, respectively. Conclusion: The CBDE test is a good alternative to broth microdilution in routine microbiology laboratories for the Enterobacterales group since it provides easy-to-apply, cost-effective testing for colistin susceptibility and has a high categorical agreement compared to the reference microdilution method. Keywords: colistin, disc elution, antibiotic resistance, broth microdilution, Enterobacterales

A prototype biospecklemeter for microbiological analysis: a starting point for a potential digital-image laser antibiotic susceptibility test

This work describes the design and construction of a low-cost prototype biospecklemeter. In this first report of its applications a microbiological analysis was performed. The relationship between microorganism density, motility and biospeckle activity was evaluated. The aim is to produce a device for research and for student use. The biospecklemeter was constructed on an antivibration table with low-cost materials and accessories and assayed using different microorganisms grown in Mueller–Hinton broth and/or agar as well as urine samples. Three lasers were installed (blue, green and red) as well as two webcams and a charge-coupled device camera. The samples were carried within the wells of a VDRL plate and 5–30 s videos or photographs of reflected light were taken. Video or photo processing was carried out using the method of temporal differences (ImageJ) or generalized differences (ImageDP). It was possible to establish a direct relationship between spectrophotometric absorption, the microbial count and the biospeckle intensity; changes in motility and the effect of antibiotics on bacterial laboratory samples as well as on urine samples were also detected. Finally, with this method colonies of different microorganisms on Mueller–Hinton agar were studied.

In vitro activity of six biocides against carbapenemase-producing Klebsiella pneumoniae and presence of genes encoding efflux pumps

IntroductionAcquisition of reduced susceptibility to biocides may contribute to the dissemination of high-risk (HR) clones of carbapenemase-producing Klebsiella pneumoniae (CP-Kp). The aim of this study was (a) to determinate the activity of biocides against CP-Kp, and (b) to analyse the relationship between biocide activity and the presence of efflux pumps. MethodsThe minimal inhibitory concentrations (MICs) of 6 biocides (sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, povidone-iodine, ethanol and triclosan) were determined in triplicate at 25°C and 37°C in Mueller-Hinton broth (MHB) and M9 minimum medium, against 17 CP-Kp isolates representing different clones (HR and no-HR), sequence-types (STs) and carbapenemases. Efflux pumps genes were detected by whole genome sequencing (MiSeq). ResultsMedian MICs were slightly higher at 37°C than at 25°C (p≤0.05), except for benzalkonium chloride, triclosan and ethanol. MIC medians were much higher in MHB than in M9, except for triclosan. No significant differences were observed in the median MICs, regarding the type of clone, ST or carbapenemase; cepA, acrAB, kpnEF and oqxAB genes were detected in all isolates, whereas qacE and qacA were not detected; smvAR, and qacΔE genes were detected in 94% and 47% of isolates, respectively. ConclusionsTriclosan, chlorhexidine digluconate, benzalkonium chloride and ethanol were the most active biocides. The activity of some biocides is affected by temperature and growth media, suggesting that standardised procedures for biocide susceptibility testing based on MIC determination are required. This activity, in terms of MICs, are not related to the type of clone, ST, carbapenemase or the presence of the efflux pump genes.

PHYTOCHEMICAL AND MICROBIOLOGICAL ASPECTS OF THE <i>NIGELLA SATIVA</i> L. HERBS STUDY

Nigella sativa L. is a promising plant object, herbal medicinal raw materials of which can be comprehensively used for the development of drugs with an antimicrobial activity.The aim of the study was to screen and compare the antimicrobial activity of water-ethanolic extractions from the Nigella sativa L. herbs with a eucalyptus tincture of as a reference preparation.Materials and methods. Chromatograms of the extracts were obtained by thin layer chromatography in the system of chloroform – ethanol – water (26:16:3). The detection of adsorption zones was carried out in daylight, in the UV light at λ=254 nm and λ=365 nm, as well as by treatment with reagents – a 3% alcohol solution of aluminum chloride and a solution of diazobenzosulfonic acid in a 20% sodium carbonate solution. The next step was to determine the minimum inhibitory concentration by the method of double serial dilutions in Mueller-Hinton nutrient broth (Bio-Rad, USA). As test cultures, the strains of the American Type Culture Collection (ATCC) microorganisms were used: Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), as well as Candida albicans (a clinical strain). Simultaneously, an experiment to establish a “negative” control was carried out. For the comparative evaluation of the studied samples activity, its activity was compared with the reference preparation with a proven antimicrobial activity – a eucalyptus tincture.Results. For all water-ethanolic extractions and the Nigella sativa L. herb tincture, the adsorption zones characteristic of flavonoids with Rf1 = 0.28, Rf2 = 0.15, Rf3 = 0.11 were revealed, and under the action of an alcoholic solution of aluminum chloride, the fluorescence of the adsorption zones was also enhanced, which indicates the phenolic nature of these compounds.In the course of the study, it was found out that all water-ethanolic extractions from the Nigella sativa L. herbs have the greatest antimicrobial effect against the Pseudomonas aeruginosa strain. When compared with the reference preparation – a eucalyptus tincture, it was notified that the specified tincture of the Nigella sativa L. herbs has an advantage in the antimicrobial activity over the strain of Pseudomonas aeruginosa – the action at the 16-fold dilution vs the 4-fold dilution. The action on the Escherichia coli and Candida albicans strains is comparable for the both tinctures.Conclusion. The obtained results of phytochemical and microbiological analyses will be used as a rationale for the introduction of antimicrobial preparations based on the Nigella sativa herbs in medical and pharmaceutical practice.

Pharmacodynamic Target Assessment and PK/PD Cutoff Determination for Gamithromycin Against Streptococcus suis in Piglets.

Gamithromycin is a long-acting azalide antibiotic that has been developed recently for the treatment of swine respiratory diseases. In this study, the pharmacokinetic/pharmacodynamic (PK/PD) targets, PK/PD cutoff, and optimum dosing regimen of gamithromycin were evaluated in piglets against Streptococcus suis in China, including a subset with capsular serotype 2. Short post-antibiotic effects (PAEs) (0.5–2.6 h) and PA-SMEs (2.4–7.7 h) were observed for gamithromycin against S. suis. The serum matrix dramatically facilitated the intracellular uptake of gamithromycin by S. suis strains, thus contributing to the potentiation effect of serum on their susceptibilities, with a Mueller-Hinton broth (MHB)/serum minimum inhibitory concentration (MIC) ratio of 28.86 for S. suis. Dose-response relationship demonstrated the area under the concentration (AUC)/MIC ratio to be the predictive PK/PD index closely linked to activity (R2 > 0.93). For S. suis infections, the net stasis, 1–log10, and 2–log10 kill effects were achieved at serum AUC24h/MIC targets of 17.9, 49.1, and 166 h, respectively. At the current clinical dose of 6.0 mg/kg, gamithromycin PK/PD cutoff value was determined to be 8 mg/L. A PK/PD-based dose assessment demonstrated that the optimum dose regimen of gamithromycin to achieve effective treatments for the observed wild-type MIC distribution of S. suis in China with a probability of target attainment (PTA) ≥ 90% was 2.53 mg/kg in this study. These results will aid in the development of clinical dose-optimization studies and the establishment of clinical breakpoints for gamithromycin in the treatment of swine respiratory infections due to S. suis.

Activity of Delafloxacin and Levofloxacin against Stenotrophomonas maltophilia at Simulated Plasma and Intrapulmonary pH Values.

ABSTRACTFluoroquinolones have become a popular treatment option for Stenotrophomonas maltophilia infections. Although levofloxacin is most commonly used, delafloxacin demonstrates comparable in vitro activity when evaluated under standard susceptibility testing conditions at neutral pH. At acidic pH, the activity of the anionic delafloxacin is improved, while the activity of the zwitterionic levofloxacin is reduced. Because the human respiratory tract has a pH of ~6.6 and is the most common site of S. maltophilia infection, it is vital to understand the activity of these agents in this environment. Therefore, levofloxacin and delafloxacin were tested against clinical S. maltophilia isolates via broth microdilution testing (n = 37) and time-kill analysis (n = 5) in neutral cation-adjusted Mueller-Hinton broth (CAMHB) (pH 7.3) and acidic CAMHB (aCAMHB) (pH 6.5). In CAMHB, MIC50 values were similar between levofloxacin and delafloxacin (8 mg/L versus 8 mg/L). In aCAMHB, levofloxacin MICs did not change, while delafloxacin MICs decreased by a median of 4 log2 dilutions (MIC50 values of 8 mg/L versus 0.25 mg/L). In time-kill analyses, levofloxacin and delafloxacin at the maximum drug concentration for the free drug (fCmax) were bactericidal against 3 and 2 isolates in CAMHB, respectively. In aCAMHB, levofloxacin was not bactericidal against any isolate, while delafloxacin was bactericidal against the same 2 isolates. Relative to CAMHB, levofloxacin activity was reduced by 2.5 log10 CFU/mL in aCAMHB, whereas delafloxacin activity was increased 2.7 log10 CFU/mL. Although the bactericidal activity of levofloxacin against S. maltophilia was attenuated in an acidic environment in this study, the increased potency of delafloxacin at pH 6.5 did not translate into improved bactericidal activity in time-kill analyses, compared to pH 7.3.IMPORTANCE Stenotrophomonas maltophilia most often infects the lungs, where the physiologic environment is naturally slightly acidic (pH ~6.6), compared to most parts of the body (such as the bloodstream), which have neutral pH values (~7.4). Pneumonia due to S. maltophilia is often treated with the antibiotic levofloxacin, despite the activity of levofloxacin being known to be impaired at acidic pH. Unfortunately, currently available methods for susceptibility testing of levofloxacin against S. maltophilia are performed at a neutral pH and therefore may not accurately represent the activity of levofloxacin at the site of infection in the lungs. A similar but newer antibiotic in the same class as levofloxacin, namely, delafloxacin, is not affected by being in an acidic environment and may actually work better at lower pH values. Therefore, the purpose of this study was to investigate whether one drug might be better than the other in this setting by testing each agent’s ability to kill S. maltophilia at pH 7.3 and pH 6.5. These findings could then be used to design confirmatory studies that may ultimately impact which drug is given to patients with lung infections due to S. maltophilia.

Phenotypic and genotypic characterisation of thymine auxotrophy in Escherichia coli isolated from a patient with recurrent bloodstream infection.

IntroductionThymine auxotrophic in vitro mutants of Escherichia coli were first reported in the mid-20th century. Later, thymine-dependent clinical strains of E. coli as well as other Enterobacterales, Enterococcus faecalis and Staphylococcus aureus have been recognized as the cause of persistent and recurrent infections.ObjectivesThe aim of this study was to characterize the phenotype and investigate the molecular basis of thymine auxotrophy in ten E. coli isolates obtained at different time points from a patient with recurrent bloodstream infection (BSI) due to a chronic aortic graft infection treated with Trimethoprim/sulfamethoxazole (TMP-SMX).MethodsClinical data was obtained from hospital records. Growth characterization and antimicrobial susceptibility testing to TMP-SMX was performed on M9 agar and in MH broth with different thymine concentrations (0.5, 2, 5, 10 and 20 μg/mL), on Mueller-Hinton (MH) and blood agar. Whole genome sequencing (WGS) was performed on all E. coli isolates.ResultsE. coli were isolated from ten consecutive BSI episodes from a patient with chronic aortic graft infection. Six of these isolates were resistant to TMP-SMX when assayed on blood agar. Growth experiments with added thymine confirmed that these isolates were thymine-dependent (thy-), and revealed growth defects (slower growth rate and smaller colony size) in these isolates relative to thy+ isolates (n = 4). WGS indicated that all isolates were of the same clonal lineage of sequence type 7358. Genomic analysis revealed a G172C substitution in thyA in all TMP-SMX resistant isolates, while mutations affecting genes involved in the deoxyribose salvage pathway (deoB and deoC) were identified in eight isolates.ConclusionThis case highlights the risk of resistance development to TMP-SMX, especially for long-term treatment, and the possible pitfalls in detection of growth-deficient subpopulations from chronic infections, which could lead to treatment failure.

Highly Synergistic Effects of Melittin With Vancomycin and Rifampin Against Vancomycin and Rifampin Resistant Staphylococcus epidermidis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) strains are increasingly emerging as serious pathogens because they can be resistant to many antibiotics called multidrug resistance (MDR) that limit the therapeutic options. In the case of vancomycin- and rifampin-resistant MDR-MRSE, the physicians are not allowed to increase the doses of antibiotics because of severe toxicity. Accordingly, we investigated the synergistic activity of melittin antimicrobial peptide with vancomycin and rifampin against vancomycin-resistant, and rifampin-resistant MDR-MRSE isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and fractional bactericidal concentration index (FBCi) of antimicrobial agents against isolates were determined. Coagulate activities and serum and salt stability as well as melittin cytotoxicity on the human embryonic kidney (HEK) 293 cells and human red blood cells (RBCs) at their synergistic concentrations. MIC and MBC values for melittin were in the range of 0.312–2.5 and 0.312–5, respectively. Results also showed that the interaction of melittin with drugs was highly synergistic in which the geometric means of FICi and FBCi were < 0.5. Induced synergism led to a decrease in melittin, rifampin, and vancomycin concentrations by 8–1,020, 2–16, and 4–16-folds, respectively. This phenomenon caused a reduction in melittin toxicity by which the synergistic concentration of melittin needed to kill bacteria did not show cytotoxicity and hemolytic activity. Besides, no coagulation activity was found for the synergistic and alone concentrations of melittin in both Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). Interestingly, the antibacterial activity of melittin in Mueller Hinton Broth (MHB) containing human serum did no significant differences between MIC and MBC values of melittin in MHB and MHB containing 10% human serum. The present findings showed that the therapeutic index of melittin was improved by 32.08- and 12.82-folds when combined with vancomycin and rifampin, respectively. Taken together, the obtained data show that melittin alone was effective against MDR-MRSE isolates and this antimicrobial peptide showed highly synergistic effects with vancomycin and rifampin without causing toxicity. Therefore, the combination of melittin and traditional antibiotics could be a promising strategy for the treatment of infections caused by MDR-MRSE.

Design, synthesis, antimicrobial activity and in silico molecular docking studies of some sulfur containing pyrazole-pyridine hybrids

We developed and synthesized novel 4-thiazolidinone hybrids with pyridine and pyrazole heterocycles (5a-5o). The structures of the synthesized products were elucidated by IR, 1H NMR and 13C NMR spectroscopy and mass spectrometry. The synthesized hybrids were investigated for their antimicrobial activity against several bacterial and fungal strains using the Mueller–Hinton broth method. Compound 5-(3-methoxybenzylidene)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-3-(pyridin-3-yl)thiazolidin-4-one (MIC = 50 μg/mL) exhibited prominent activity against gram-negative E. coli while compounds 5-(2-methylbenzylidene)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-3-(pyridin-3-yl)thiazolidin-4-one and 5-(4-methylbenzylidene)-2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-3-(pyridin-3-yl)thiazolidin-4-one exhibited MIC value of 250 μg/mL against C. albicans. Molecular docking study against microbial DNA gyrase could provide valuable insights into the binding modes of these molecules. They were found to exhibit excellent binding affinity (average Glide docking score: −7.989 and binding energy: −49.807 kcal/mol) through significant bonded and non-bonded interactions.

Zinc Oxide and Tea Theaflavin Suppress Enterotoxigenic Salmonella Isolated From Swine Livestock

Objectives Salmonella infection can lead to diarrhea and diminished weight gains in livestock, as well as gastroenteritis in humans. Although antibiotics have been used historically on the farm to promote animal growth, this practice may also foster development of resistant bacterial strains and thereby deplete our arsenal of effective antibiotic therapies. Here, Salmonella typhimurium (isolated from cecal contents of swine) is shown to be vulnerable to zinc oxide (ZnO) and theaflavin 3,3'-digallate (TF3), each at gastrointestinal concentrations achievable via the diet.MethodsPropagation of Salmonella in Mueller-Hinton broth suspension was assessed in dose-response experiments involving ZnO or TF3 exposures, and subsequently in experiments using a combination of these compounds.ResultsThe minimum effective concentrations against 6-hour growth of Salmonella at 37°C were 1 mM ZnO (62% inhibition) or 25 μM TF3 (30% inhibition). Salmonella growth was inhibited up to 99.6% when exposed to 7 mM ZnO, and up to 89% when exposed to 100 μM TF3. In concurrent experiments assessing individual effects of either ZnO or TF3, as well as a combination of these treatments, the minimum effective concentration of ZnO was found to be more potent given alone than when given in combination with TF3.ConclusionsThese findings suggest that physiological concentrations of ZnO within the human gut lumen (upon consuming dietary supplements containing less than the Tolerable Upper Intake Level) may limit or prevent the ability of Salmonella to propagate. Similarly, TF3 (at human gut concentrations upon consuming less than a single cup of black tea) appears to suppress Salmonella, yet TF3 could compete or otherwise constrain the efficacy of ZnO. Applications of these findings may include customized ZnO or TF3 delivery platforms for use in humans at high risk of diarrheal disease or other gastrointestinal disorders (e.g., military personnel on deployment under austere environmental conditions), as well as in agricultural practices to support animal welfare and normal growth without need for therapeutic antibiotics.Funding SourcesSupported by USDA and Texas A&M AgriLife Research.

Improvement of antimicrobial susceptibility testing in biofilm-growingcoagulase-negative Staphylococcus hominis

Coagulase-negative Staphylococcus hominis causes bloodstream infections and often can form biofilms on medical devices. This study aimed to improve the current methodology for antimicrobial susceptibility testing (AST) in biofilm-growing S. hominis isolates. Biofilm production of S. hominis was assessed using the crystal violet staining method in trypticase soy broth supplemented with 1% glucose (TSBglu1%), Mueller-Hinton broth (MHB), or MHBglu1% using flat-bottom plates or the Calgary device. Susceptibility to antibiotics was assessed using the broth microdilution method (MHB and TSBglu1%) in planktonic cells (round-bottom plates) and biofilm cells (flat-bottom plates and the Calgary device). Biofilm determination using TSBglu1% yielded better performance over MHB, and flat-bottom plates without agitation were preferred over the Calgary device. Higher fold dilution values between the minimum biofilm eradication concentration (MBEC) and the minimum inhibitory concentration (MIC) were obtained in MHB for almost all antibiotics, except for linezolid. TSBglu1% and flat-bottom polystyrene plates were preferred over MHB and the Calgary device for biofilm determination. AST in biofilm-growing S. hominis showed better performance using TSBglu1% compared to MHB. Therefore, when comparing MBEC and MIC values, AST in planktonic cells could also be performed using TSBglu1% instead of MHB.