Note. In Vitro Viability of Bifidobacterium Strains Isolated from Commercial Dairy Products Exposed to Human Gastrointestinal Conditions

2005 ◽  
Vol 11 (4) ◽  
pp. 307-314 ◽  
Author(s):  
M. C. Collado ◽  
Y. Moreno ◽  
E. Hernández ◽  
J. M. Cobo ◽  
M. Hernández
Keyword(s):  
Dairy Products ◽  
Viable Cell ◽  
Human Gastrointestinal Tract ◽  
Cell Counts ◽  
Gastrointestinal Conditions ◽  
Acid Tolerant ◽  
Viable Bacteria ◽  
Selection Of ◽  
Direct Counts

Levels of bifidobacteria contained in commercial fermented milks in Spain were determined by fluorescent techniques. The transit tolerance of probiotic bifidobacteria strains to human gastrointestinal tract (GIT) was assessed in vitro. The number of bifidobacteria in commercial fermented milks declared to contain bifidobacteria varied from 104 to 107 bacteria/m L. Viable cell counts estimated by plating onto selective media were lower than direct counts. Bifidobacteriumstrains analysed showed different survival behaviour. Viable bacteria counts decreased considerably following exposure to gastric juice. As only intrinsic acid resistant cells survive their passage through the human intestine, the selection of acid tolerant strains is necessary for the elaboration of probiotic products. Viability of dairy bifidobacteria is affected by gastrointestinal juices but the majority of tested strains survived well at gastrointestinal conditions. The reason for this may be the low number of viable bifidobacteria contained in commercial dairy products. Adaptation and survival at low pH is likely to determine the efficacy of Bifidobacterium strains both as dairy starters and probiotic microorganisms. This study confirmed the usefulness of fluorescent techniques for a rapid and accurate evaluation of bacterial viability in probiotic products.

scholarly journals 1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S643-S643
Author(s):  
Maria F Mojica ◽  
Christopher Bethel ◽  
Emilia Caselli ◽  
Magdalena A Taracila ◽  
Fabio Prati ◽  
...  
Keyword(s):  
Active Site ◽  
Inhibitory Activity ◽  
Covalent Bond ◽  
Thiol Group ◽  
Viable Cell ◽  
Free Thiol ◽  
Transition State Analogs ◽  
Cell Counts ◽  
Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

In Vitro Anti-Biofilm Activity of Hydrogen-Peroxide Generating Electrochemical Bandage Against Yeast Biofilms

2021 ◽  
Author(s):  
Yash S. Raval ◽  
Abdelrhman Mohamed ◽  
Jayawant N. Mandrekar ◽  
Cody Fisher ◽  
Kerryl E. Greenwood-Quaintance ◽  
...  
Keyword(s):  
Membrane Damage ◽  
Microscopic Analysis ◽  
Viable Cell ◽  
Wound Infections ◽  
Unique Challenge ◽  
Cell Membrane Damage ◽  
Cell Counts ◽  
Extensive Cell ◽  
Fluorescence Microscopic Analysis

Wound infections are caused by bacteria and/or fungi. The presence of fungal biofilms in wound beds presents a unique challenge, as fungal biofilms may be difficult to eradicate. The goal of this work was to assess the in vitro anti-biofilm activity of a H 2 O 2 -producing electrochemical bandage (e-bandage) against 15 yeast isolates representing commonly-encountered species. Time-dependent decreases in viable biofilm CFU counts of all isolates tested were observed, resulting in no visible colonies with 48 hours of exposure by plate culture. Fluorescence microscopic analysis showed extensive cell membrane damage of biofilm cells after e-bandage treatment. Reductions in intracellular ATP levels of yeast biofilm cells were recorded post e-bandage treatment. Our results suggest that exposure to H 2 O 2 -producing e-bandages reduce in vitro viable cell counts of yeast biofilms, making this a potential new topical treatment approach for fungal wound infections.

scholarly journals NCOA4 Mediates Ferritin Responses to Iron, Erythrophagocytosis, and Hepcidin in Macrophages (P24-056-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Cole Guggisberg ◽  
Moon-Suhn Ryu
Keyword(s):  
Iron Homeostasis ◽  
Primary Source ◽  
Viable Cell ◽  
Iron Chelator ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Anemia Of Chronic Disease ◽  
Cell Counts ◽  
Subsequent Decrease

Abstract Objectives Iron recycled from erythrophagocytosis by macrophages serves as a primary source of systemic iron. NCOA4 mediates ferritin turnover via ferritinophagy. Yet, whether NCOA4 is important in macrophages or erythrophagocytosis-mediated iron recycling remains unclear, and thus was assessed in vitro. Methods J774 cells were employed as an in vitro model of macrophages. Iron studies involved treatments of ferric ammonium citrate (FAC) or an iron chelator, deferoxamine (Dfo). To recapitulate systemic iron recycling and overload, cells were treated with opsonized erythrocytes and minihepcidin, respectively. NCOA4 knock-down was achieved by siRNA transfection. Iron gene responses were measured by qPCR and western analyses, and viable cell counts were colorimetrically determined by CCK8 assays as functional outcomes. Results NCOA4 protein abundance was inversely related to iron availability and ferritin in macrophages. Loss of NCOA4 resulted in impaired ferritin turnover, and led to a reduction in viable cells when combined with iron deficiency. By erythrophagocytosis, a peak in ferritin abundance was observed at 12 h with a subsequent decrease at 24 h. This loss in ferritin was NCOA4-dependent. Minihepcidin caused accumulation of ferritin, along with a repression of NCOA4 in both control and erythrocyte-laden macrophages. Hepcidin activity had no effect on ferritin when NCOA4 was depleted. Conclusions NCOA4 mediates the release of ferritin iron during cellular iron restriction and iron recycling by macrophages. Moreover, our studies suggest that macrophage NCOA4 is integral to systemic iron homeostasis by responding to the iron regulatory hormone, hepcidin. Thus, NCOA4 and ferritinophagy may potentially serve as therapeutic targets for treatments of iron disorders and anemia of chronic disease. Funding Sources Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.

scholarly journals Activity of pyrazinamide in a murine model against Mycobacterium tuberculosis isolates with various levels of in vitro susceptibility.

1996 ◽  
Vol 40 (1) ◽  
pp. 14-16 ◽  
Author(s):  
S P Klemens ◽  
C A Sharpe ◽  
M H Cynamon
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Efficacy ◽  
Viable Cell ◽  
Test System ◽  
Infection Model ◽  
Drug Resistant ◽  
In Vitro Susceptibility ◽  
Cell Counts ◽  
Treatment Of Infection

The activity of pyrazinamide (PZA) against eight isolates of Mycobacterium tuberculosis in a murine infection model was evaluated. M. tuberculosis isolates with various degrees of in vitro susceptibility to PZA (MIC range, 32 to > 2,048 micrograms/ml) were used. Four-week-old female mice were infected intravenously with approximately 10(7) viable M. tuberculosis organisms. PZA at 150 mg/kg of body weight was started 1 day postinfection and given 5 days/week for 4 weeks. Infected but untreated mice were compared with PZA-treated mice. Mice were sacrificed at the completion of the treatment period, and viable cell counts were determined from homogenates of spleens and right lungs. PZA had activity in the murine test system against M. tuberculosis isolates for which the MICs were < or = 256 micrograms/ml. However, there was an inconsistent correlation between the absolute MICs and the reductions in organ viable cell counts. Studies with drug-resistant M. tuberculosis isolates with an isogenic background would improve evaluation of drug efficacy in the murine test system. Further evaluation of antimycobacterial agents against monodrug-resistant isolates will provide data that will be useful for development of algorithms for treatment of infection with drug-resistant organisms.

scholarly journals An Integrated HOCl-Producing E-Scaffold Is Active Against Monomicrobial and Polymicrobial Biofilms

2021 ◽  
Author(s):  
Laure Flurin ◽  
Yash S. Raval ◽  
Abdelrhman Mohamed ◽  
Kerryl E. Greenwood-Quaintance ◽  
Edison J. Cano ◽  
...  
Keyword(s):  
Low Dose ◽  
Hypochlorous Acid ◽  
Viable Cell ◽  
Chloride Ions ◽  
Resistant Bacteria ◽  
Wound Infections ◽  
Microbial Biofilms ◽  
Cell Counts ◽  
Acquired Antibiotic Resistance

Oxidizing agents like hypochlorous acid (HOCl) have antimicrobial activity. We developed an integrated electrochemical scaffold or ‘e-scaffold’ that delivers a continuous low dose of HOCl aimed at targeting microbial biofilms without exceeding concentrations toxic to humans, as a prototype of a device being developed to treat wound infections in humans. In this work, we tested the device against 33 isolates of bacteria (including isolates with acquired antibiotic resistance) grown as in vitro biofilms, alongside 12 combinations of dual-species in vitro biofilms. Biofilms were grown on the bottoms of 12-well plates for 24 hours. An integrated e-scaffold was placed atop each biofilm and polarized at 1.5V for 1, 2 or 4 hours. HOCl was produced electrochemically by oxidizing chloride ions (Cl-) in solution to chlorine (Cl2); dissolved Cl2 spontaneously dissociates in water to produce HOCl. The cumulative concentration of HOCl produced at the working electrode in each well was estimated to be 7.89, 13.46, and 29.50 mM after 1, 2 and 4 hours of polarization, respectively. Four hours of polarization caused an average reduction of 6.13 log10 CFU/cm2 (±1.99 log10 CFU/cm2) of viable cell counts of mono-species biofilms and 5.53 log10 CFU/cm2 (±2.31 log10 CFU/cm2) for the 12 dual-species biofilms studied. The described integrated e-scaffold reduces viable bacterial cell counts in biofilms formed by an array of antibiotic-susceptible and -resistant bacteria alone and in combination.

Inhibitory Effects of Enterococcus Strains Obtained from a Probiotic Product on In Vitro Growth of Salmonella enterica Serovar Enteritidis Strain IFO3313

2006 ◽  
Vol 69 (9) ◽  
pp. 2258-2262 ◽  
Author(s):  
WATTHANA THEPPANGNA ◽  
KOICHI OTSUKI ◽  
TOSHIYUKI MURASE
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enteritidis ◽  
Viable Cell ◽  
Mixed Cultures ◽  
Inhibitory Effects ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Cell Counts ◽  
Pure Cultures ◽  
Probiotic Product

Enterococcus faecium and Enterococcus gallinarum strains were isolated from a commercial probiotic product and the effects of these strains on the growth of Salmonella enterica serovar Enteritidis strain IFO3313 were investigated. Viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. faecium were significantly (P &lt; 0.05) lower than those in pure cultures after 6, 8, and 24 h when the cultures were incubated in heart infusion broth at 37 and 41°C. Significant differences in viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. gallinarum and those in pure cultures were also observed after 8 and 24 h at 37 and 41°C. Similar observations were shown in mixed cultures of Salmonella Enteritidis with the reference strains of E. faecium GIFU8355 and E. gallinarum ATCC 49573. Significant differences in viable cell counts of these enterococcal strains were not shown among pure and mixed cultures with Salmonella Enteritidis. The pH values in pure and mixed cultures were 7.0 or 7.5 throughout the experiments. E. faecium strains were found to harbor the genes encoding enterocins A and B and showed inhibitory zones with a diameter of 4 to 6 mm against growth of Salmonella Enteritidis in the enterocin production assays. However, the E. gallinarum strains possessed neither of the enterocin genes tested and exhibited no inhibition zone in the enterocin production assays. These results indicated that enterococcal strains exhibit inhibitory effects on the growth of Salmonella Enteritidis and these effects were due to both enterocin and nonenterocin factors.

scholarly journals Combination Therapy with Disulfiram, Copper, and Doxorubicin for Osteosarcoma: In Vitro Support for a Novel Drug Repurposing Strategy

Sarcoma ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Jonathan B. Mandell ◽  
Feiqi Lu ◽  
Matthew Fisch ◽  
Jan H. Beumer ◽  
Jianxia Guo ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Efflux Pump ◽  
Copper Chloride ◽  
Drug Repurposing ◽  
Viable Cell ◽  
Metastatic Potential ◽  
Trypan Blue Exclusion ◽  
Cell Counts ◽  
Triple Treatment

Although many cancer cells have significantly higher copper concentrations compared with normal cells and tissues, the role of copper in cancer biology and metastatic disease remains poorly understood. Here, we study the importance of copper in osteosarcoma, which frequently metastasizes to the lungs and is often chemoresistant. K12 and K7M2 are murine OS cells with differing metastatic phenotypes: K7M2 is highly metastatic, whereas K12 is much less so. Intracellular copper levels were determined using atomic absorption. Copper transporters were quantified by qPCR. Cytotoxicity of doxorubicin, disulfiram, and copper(II) chloride was assessed with a cell viability fluorescence stain. Additionally, K7M2 viable cell counts were determined by trypan blue exclusion staining after 72 hours of treatment. Copper levels were found to be significantly higher in K12 OS cells than in K7M2 cells. qPCR showed that K12 cells upregulate the copper influx pump CTR1 and downregulate the copper efflux pump ATP7A compared to K7M2 OS cells. Combination treatment of copper chloride (50 nM) with disulfiram (80 nM) was only cytotoxic to K12 cells. Triple treatment with doxorubicin, disulfiram, and copper displayed potent and durable cytotoxicity of highly metastatic K7M2 cells. We demonstrate here that murine OS cell lines differing in metastatic potential also vary in endogenous copper levels and regulation. Additionally, these differences in copper regulation may contribute to selective cytotoxicity of K12 cells by extremely low doses of copper-potentiated disulfiram. The combination of doxorubicin, disulfiram, and copper should be explored as a therapeutic strategy against OS metastases.

scholarly journals Validation of a Noninvasive, Real-Time Imaging Technology Using Bioluminescent Escherichia coli in the Neutropenic Mouse Thigh Model of Infection

2001 ◽  
Vol 45 (1) ◽  
pp. 129-137 ◽  
Author(s):  
H. L. Rocchetta ◽  
C. J. Boylan ◽  
J. W. Foley ◽  
P. W. Iversen ◽  
D. L. LeTourneau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Antimicrobial Agents ◽  
Viable Cell ◽  
Multicopy Plasmid ◽  
In Vivo Evaluation ◽  
Therapeutic Success ◽  
Cell Counts

ABSTRACT A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. Thelux gene cluster of Photorhabdus luminescenswas introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

scholarly journals Vancomycin Acts Synergistically with Gentamicin against Penicillin-Resistant Pneumococci by Increasing the Intracellular Penetration of Gentamicin

2003 ◽  
Vol 47 (1) ◽  
pp. 144-147 ◽  
Author(s):  
P. Cottagnoud ◽  
M. Cottagnoud ◽  
M. G. Täuber
Keyword(s):  
Viable Cell ◽  
Underlying Mechanism ◽  
Intracellular Concentration ◽  
Combination Regimen ◽  
Cell Counts ◽  
Short Period ◽  
Before And After ◽  
Gentamicin Concentration

ABSTRACT Vancomycin and gentamicin act synergistically against penicillin-resistant pneumococci in vitro and in experimental rabbit meningitis. The aim of the present study was to investigate the underlying mechanism of this synergism. The intracellular concentration of gentamicin was measured by using the following experimental setting. Bacterial cultures were incubated with either gentamicin alone or gentamicin plus vancomycin for a short period (15 min). The gentamicin concentration was determined before and after grinding of the cultures by using the COBAS INTEGRA fluorescence polarization system (Roche). The grinding efficacies ranged between 44 and 54%, as determined by viable cell counts. In the combination regimen the intracellular concentration of gentamicin increased to 186% compared to that achieved with gentamicin monotherapy. These data suggest that the synergy observed in vivo and in vitro is based on an increased intracellular penetration of the aminoglycoside, probably due to the effect of vancomycin on the permeability of the cell wall.

scholarly journals Antilisterial Effects of Gravinol-S Grape Seed Extract at Low Levels in Aqueous Media and Its Potential Application as a Produce Wash

2010 ◽  
Vol 73 (2) ◽  
pp. 266-273 ◽  
Author(s):  
BLEDAR BISHA ◽  
NATALIA WEINSETEL ◽  
BYRON F. BREHM-STECHER ◽  
AUBREY MENDONCA
Keyword(s):  
Antimicrobial Agents ◽  
Aqueous Media ◽  
Membrane Integrity ◽  
Viable Cell ◽  
Grape Seed Extract ◽  
Grape Seed ◽  
Seed Extract ◽  
Live Cells ◽  
Cell Counts

Grape seed extract (GSE) is a rich source of proanthocyanidins, a class of natural antioxidants reported to have wide-ranging bioactivity as anti-inflammatory, anticarcinogenic, and antimicrobial agents. The ability of GSE to rapidly inactivate Listeria monocytogenes in vitro and the generally recognized as safe status of GSE make this extract an attractive candidate for control of Listeria in or on foods. Previously, GSE has been used at relatively high concentrations (1%) in complex food matrices and in combination with other antimicrobials. We sought to characterize the antilisterial effects of a commercial GSE preparation (Gravinol-S) alone at much lower concentrations (0.00015 to 0.125%) in aqueous solution and to test its possible use as an antimicrobial wash for fresh produce surfaces. Based on broth microdilution tests, the MICs of GSE against L. monocytogenes Scott A and Listeria innocua ATCC 33090 were as low as 50 and 78 μg ml−1, respectively. GSE was evaluated in 0.85% saline against live cells of L. innocua via flow cytometry, using propidium iodide as a probe for membrane integrity. At sub-MICs and after only 2 min of exposure, treatment with GSE caused rapid permeabilization and clumping of L. innocua, results that we confirmed for L. monocytogenes using fluorescence microscopy and Live/Dead staining. At higher concentrations (0.125%), GSE reduced viable cell counts for L. monocytogenes by approximately 2 log units within 2 min on tomato surfaces. These results suggest the potential for GSE as a natural control of Listeria spp. on low-complexity foods such as tomatoes.

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