Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins

Kaspar, Ursula; Schleimer, Nina; Idelevich, Evgeny A.; Molinaro, Sonja; Becker, Karsten

doi:10.3390/microorganisms10020445

Cited by 4 publications

(5 citation statements)

References 28 publications

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“…The rapid concentration-dependent bactericidal effect of exebacase was in contrast to the time-dependent cidal activity of vancomycin and oxacillin, two conventional antibiotics that interfere with peptidoglycan biosynthesis, and which showed only about 1-log 10 CFU/mL bactericidal reduction over 3 h, consistent with other reports ( 26 , 27 ). Regrowth at 24 h was observed with exebacase for all isolates in the time-kill assays, a reported phenomenon with lysins ( 21 ). This regrowth is likely due to the very rapid mode of action for lysins, wherein large amounts of simultaneously released cellular debris enclose occasional surviving staphylococci, which are protected from the lysin and subsequently allow for regrowth.…”

Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

“…Regrowth following a bactericidal effect in the time-kill assay format is commonly observed with lysins ( 21 ) and is believed to arise from rapid “overkilling,” whereby cellular debris encases some staphylococci and provides a protective effect that later enables regrowth as lysin concentrations decline through binding to cellular debris. This effect is likely limited to in vitro conditions ( 21 ). Following CLSI recommendations for regrowth observed in time-kills ( 17 ), exebacase MICs were determined for survivors and shown to be identical to that of isolates prior to treatment; thus, survival was not be attributed to resistance.…”

Section: Resultsmentioning

confidence: 99%

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Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase

Vila-Farrés

Sauve

et al. 2023

Microbiol Spectr

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Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among pathogenic bacteria. Exebacase is an antistaphylococcal lysin and the first member of the lysin class to have entered clinical trials in the United States. In this study, the bacteriolytic activity of exebacase was characterized with time-kill assays, turbidity reduction assays, and microscopy. Three methicillin-susceptible Staphylococcus aureus and three methicillin-resistant S. aureus isolates were tested in time-kill assays over a range of concentrations from 0.25 to 8 × MIC. Exebacase demonstrated a concentration-dependent killing and showed bactericidal activity (≥3 log 10 kill achieved relative to the starting inoculum) within 3 h at 1 × MIC against all strains tested. Dose-dependent lysis by exebacase was, furthermore, observed in the turbidity reduction assay, wherein decreases in initial OD 600 of 50% were observed within ~15 min at concentrations as low as 4 µg/mL. Membrane dissolution, loss of cytoplasmic material, and lysis were confirmed by video and electron microscopy. The demonstrated rapid bacteriolytic effect of exebacase is an important distinguishing feature of this novel modality. IMPORTANCE To guide the development of an investigational new antibacterial entity, microbiological data are required to evaluate the killing kinetics against target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus . Killing by exebacase was determined in multiple assay formats including time-kill assays, wherein reductions of viability of ≥3 log 10 colony-forming units/mL were observed within 3 h for multiple different isolates tested, consistent with very rapid bactericidal activity. Rapid reductions in optical density were likewise observed in exebacase-treated cultures, which were visually consistent with microscopic observations of rapid lysis. Overall, exebacase provides a novel antimicrobial modality against S. aureus , characterized by a rapid cidal and lytic activity.

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Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase

Vila-Farrés

Sauve

et al. 2023

Microbiol Spectr

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“…In this first study of sodium bituminosulfonate’s bactericidity, a very rapid bactericidal effect against MSSA and MRSA was observed, with 99.9% of the inoculum already killed after a short incubation time of 30 min by concentrations corresponding to 16× MIC and 256× MIC ( Figure 1 ). Regrowth—a phenomenon occasionally observed with the time-kill methodology [ 31 , 32 ]—was noted at lower concentrations of 1× MIC, 2× MIC and 4× MIC, while the higher concentrations of 16× MIC and 256× MIC caused irreversible killing. In conclusion, a bactericidal effect was demonstrated for sodium bituminosulfonate by MBC determination on a clinical collection of MSSA and MRSA strains.…”

Section: Discussionmentioning

confidence: 98%

Bactericidal Activity of Sodium Bituminosulfonate against Staphylococcus aureus

2022

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Antibiotic resistance is increasing worldwide making it necessary to search for alternative antimicrobials. Sodium bituminosulfonate is a long-known substance, whose antimicrobial inhibitory activity has recently been re-evaluated. However, to the best of our knowledge, the bactericidal mode of action of this substance has not been systematically characterized. The aim of this study was to investigate the in vitro bactericidal activity of sodium bituminosulfonate by determining the minimal bactericidal concentrations (MBC), as well as the rapidity of bactericidal effect by time-kill curves. Clinical isolates of methicillin-susceptible (MSSA, n = 20) and methicillin-resistant (mecA/mecC-MRSA, n = 20) Staphylococcus aureus were used to determine MBC by a broth microdilution method. Sodium bituminosulfonate (Ichthyol® light) was tested in double-dilution concentration steps ranging from 0.03 g/L to 256 g/L. For time-kill analysis, two reference and two clinical S. aureus strains were tested with different concentrations of sodium bituminosulfonate (1× minimal inhibitory concentration (MIC), 2× MIC, 4× MIC, 16× MIC and 256× MIC). For MSSA isolates, MBC50, MBC90 and the MBC range were 0.5 g/L, 1.0 g/L and 0.125–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 4, 4 and 1–8, respectively. Among MRSA isolates, MBC50, MBC90 and the MBC range amounted to 0.5 g/L, 1.0 g/L and 0.06–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 2, 4 and 1–8, respectively. Time-kill kinetics revealed a bactericidal effect after 30 min for sodium bituminosulfonate concentrations of 16× MIC and 256× MIC. The bactericidal activity against MSSA and MRSA was demonstrated for sodium bituminosulfonate. The killing was very rapid with the initial population reduced by 99.9% after only short incubation with concentrations of 16× MIC and higher.

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“…Subsequently, 1 ml of the inoculum was added. Further samples were taken from each tube to monitor by pour plate count at different time intervals (0, 2, 4, 6, 8, and 12 h) 28 .…”

Section: Determination Of Bacterial Time-kill Curvementioning

confidence: 99%

The dual activity of CaONPs as a cancer treatment substance and at the same time resistance to harmful microbes

Awaad,

Olama,

El-Subruiti

et al. 2023

Sci Rep

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Nanotechnology holds significant promise for the development of novel and necessary products that enhance human health. Pharmacology and nanotechnology have contributed to developing advanced and highly effective drugs for cancer treatment and combating microbial infections. The microbiological effectiveness against the variety of examined microorganisms was assessed using the time killer curve, scanning electron microscopy (SEM), MIC techniques, and the agar well diffusion method. SEM was utilized to enhance the analysis of the mechanisms underlying the bio-interface interaction and intracellular localization of calcium oxide nanoparticles (CaONPs). The MTT test was used to examine the cytotoxicity of CaONP anticancer activity in various cancer cells, including colon, breast, and hepatic cells. The efficacy of CaONPs as an anticancer medication was elucidated by analyzing the gene expression of both treated and untreated cancer cells. MIC and MBC of CaONPs against Escherichia coli and Staphylococcus epidermidis were 150, 150, 150, and 200 µg/ml, respectively. The MIC and MFC of CaONPs against Candida albicans were 200 µg/ml and 250 µg/ml, respectively. The IC50 values of various CaONPs vary depending on the type of cancer cells. The gene expression analysis of breast cancer cells undergoing treatment revealed the identification of several cancer-controlling genes, namely BAX, BCL2, P53, TERT, KRAS1, KRAS2, and RB1. The study demonstrated the notable antibacterial efficacy of CaONPs, highlighting their potential as cancer therapies.

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Exploration of Bacterial Re-Growth as In Vitro Phenomenon Affecting Methods for Analysis of the Antimicrobial Activity of Chimeric Bacteriophage Endolysins

Cited by 4 publications

References 28 publications

Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase

Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase

Bactericidal Activity of Sodium Bituminosulfonate against Staphylococcus aureus

The dual activity of CaONPs as a cancer treatment substance and at the same time resistance to harmful microbes

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