Bacterial Phagocytosis Monitored by Fluorescence and Extracellular Quenching: Ingestion and Intracellular Killing

Goldner, M.; Farkas-Himsley, Hannah; Kormendy, Agnes C.; Skinner, Martin

doi:10.1093/labmed/14.5.291

Cited by 55 publications

(23 citation statements)

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“…Intracellular viable bacteria were determined by standard colony counts (CFU) and by a sensitive acridine orange fluorescence method in which crystal violet was used to quench the fluorescence of noningested organisms (9,10,19). Acridine orange-labeled bacteria fluoresce apple green if they are viable and brick red if they are nonviable.…”

Section: P-lactams Enhance Host Defense Mechanismsmentioning

confidence: 99%

Enhanced phagocytosis, killing, and serum sensitivity of Escherichia coli and Staphylococcus aureus treated with sub-MICs of imipenem

Adinolfi

Bonventre

1988

Antimicrob Agents Chemother

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The influence of pretreatment of Escherichia coi and Staphylococcus aureus with sub-MICs of the new P-lactam antibiotic imipenem on phagocytosis and kffling by murine peritoneal macrophages and the susceptibility of these organisms to serum bactericidal activity were studied. The effects of imipenem, a round form inducer in gram-negative rods, and piperacillin, a filamentous form inducer, were compared. Bacteria grown in the presence of sub-MICs of imipenem or piperacillin were incubated for 30 min with macrophage monolayers in the absence of antibiotic. Phagocytosis, killing, and survival within macrophages were evaluated by microbiological and fluorescence microscope assays. Bacteria grown in the presence of a sub-MIC of imipenem were phagocytized and killed in numbers significantly higher than untreated or piperacillin-treated bacteria were. Intracellular bacteria pretreated with a sub-MIC of imipenem were also readily killed by lymphokine-activated macrophages. Prior treatment with a sub-MIC of imipenem resulted in an increased susceptibility ofE. coli but not S. aureus to the bactericidal activity of immune serum. Imipenem treatment and immune serum acted synergistically to enhance phagocytosis and killing. The data indicate that exposure of E. coli and S. aureus to a sub-MIC of imipenem enhances the susceptibility of these potential pathogens to cellular and humoral host defense mechanisms.The activity of antibiotics against clinically significant bacteria is usually expressed in terms of concentrations that either inhibit growth or kill microorganisms in vitro. Concentrations of antibiotics which are less than the MIC are defined as sub-MICs (18). Evidence is accumulating that antibiotics may be beneficial to infected hosts even when present at sub-MICs (8,15,23). This effect appears to be independent of the bactericidal potential of the antibiotic but is related to structural or metabolic changes which augment the susceptibility of bacterial pathogens to humoral and cellular defenses. Therefore, antibiotics at sub-MICs that act in concert with host defenses may be clinically significant.We undertook this study to determine whether pretreatment of Escherichia coli and Staphylococcus aureus with sub-MICs of imipenem altered their susceptibility either to serum bactericidal activity or to phagocytosis and killing by murine macrophages. Imipenem, a P-lactam antibiotic and member of a new class, the carbapenems, is the first clinically available derivative of thienamycin (11). Imipenem possesses an unusually broad antibacterial spectrum, is biologically active at extremely low concentrations, and exhibits no cross resistance with other P-lactam antibiotics (13). Comparative studies indicate that there is a greater potency and wider spectrum of activity for imipenem than for other P-lactams against both gram-positive and gramnegative species (12). Another interesting attribute of imipenem is that at sub-MICs, the drug fails to induce the filamentation of gram-negative bacteria, as is the case with other penici...

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Section: P-lactams Enhance Host Defense Mechanismsmentioning

confidence: 99%

Enhanced phagocytosis, killing, and serum sensitivity of Escherichia coli and Staphylococcus aureus treated with sub-MICs of imipenem

Adinolfi

Bonventre

1988

Antimicrob Agents Chemother

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“…After staining with acridine orange, and in order to avoid overestimating phagocytosis because of the bacteria attached to the PMN surface and not yet internalized, the technique of Hed [19] and Goldner et al [20] was used to quench extracellular membrane-adherent microorganism fluorescence by means of crystal violet (500 Ìg/ml for 20 s). Since crystal violet does not penetrate PMNs, it does not alter the fluorescence of ingested microorganisms.…”

Section: Effects On Phagocytosis and Bacterial Killingmentioning

confidence: 99%

Effect of Butylated Hydroxyanisole and Some of Its Derivatives on Human Neutrophil Oxidative Burst: Chemiluminescence Evaluation

et al. 2003

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An acute inflammatory response begins during the reperfusion phase following an ischemic insult in which polymorphonuclear neutrophils (PMNs) play an important role and the release of reactive oxygen species (ROS) causes further damage and a reduction in endogenous antioxidant storage. The ability of butylated hydroxyanisole (BHA) and some phenolic, aliphatic and aromatic BHA derivatives to reduce the human PMN oxidative burst evoked by particulate (Candida albicans and zymosan) or soluble stimulants [N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA)] was investigated using luminol-amplified chemiluminescence. BHA and the derivative dt-BHA [3,5-di-t-butyl-4-hydroxyanisole] significantly reduced the PMN oxidative burst at concentrations ranging from 5 × 10^–6 to 5 × 10^–5 mol/l for C. albicans stimulation, while for zymosan stimulation, reduction was seen at concentrations ranging from 5 × 10^–6 to 5 × 10^–5 mol/l for BHA, and at concentrations ranging from 5 × 10^–7 to 5 × 10^–5 mol/l for dt-BHA, with dt-BHA being the most active. Another BHA derivative, Bu GAM 1, was active at 5 × 10^–5 mol/l for C. albicans and at 5 × 10^–6 to 5 × 10^–5 mol/l for zymosan. The findings obtained with fMLP and PMA were very similar to those previously reported. ROS release is related to PMN killing activity, but the inhibition of the PMN oxidative burst induced by BHA and BHA derivatives did not significantly modify PMN phagocytosis or killing. It has recently been observed that dt-BHA has a spasmolytic action by inhibiting the influx of Ca²⁺ into cells through L-type Ca²⁺ channels, which means that a single molecule is capable of counteracting two major steps in the sequence of events triggered by ischemia-reperfusion injury, i.e. free radical release and Ca²⁺ overload.

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“…After staining with acridine orange in order to avoid the overestimation of phagocytosis due to the E. coli attached to PMN surface and not yet internalized, the technique of Hed [11] and Goldner et al [12] was adopted to quench the extracellular membraneadherent microorganism fluorescence by crystal violet (500 Ìg/ml for 20 s). Since crystal violet does not penetrate PMNs intracellulary, it does not alter the fluorescence of ingested microorganisms.…”

Section: Phagocytosis and Bacterial Killingmentioning

confidence: 99%

Influence of Sub-Minimum Inhibitory Concentrations of Cefodizime on the Phagocytosis, Intracellular Killing and Oxidative Bursts of Human Polymorphonuclear Leukocytes

Braga

Sasso²,

Mancini³

et al. 1999

Chemotherapy

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It is generally recognized that sub-minimum inhibitory concentrations (sub-MICs) of antibiotics are still capable of interfering with some bacterial virulence parameters, thus facilitating host neutrophilic defenses such as phagocytosis, killing and oxidative bursts. This study investigated the interaction of Escherichia coli with these neutrophilic functions after pretreatment with various sub-MICs of cefodizime. E. coli exposed to 1/2 to 1/8 MICs of cefodizime showed the extensive production of long and very long filaments that interfere with the precise measurement of phagocytic and killing parameters. Our analysis was consequently extended to the activity of 1/16 to 1/64 MICs. The interesting finding was that, although phagocytosis was unaffected, killing was significantly increased in one strain at 1/16 MIC and in another at 1/32 MIC while in the last strain it was unaffected. Oxidative bursts were not modified by any of the sub-MICs. The knowledge that these sub-MICs are still effective in increasing bacteria killing, correlated with the pharmacokinetic curve of a common single dose of cefodizime 1 g i.m., showed that the killing effects of sub-MICs may last for as long as 12 h after the activity of the MIC value. This integrated information extends our knowledge of the ultimate efficacy of an antibiotic and provides further information for optimizing scheduling.

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Bacterial Phagocytosis Monitored by Fluorescence and Extracellular Quenching: Ingestion and Intracellular Killing

Cited by 55 publications

References 0 publications

Enhanced phagocytosis, killing, and serum sensitivity of Escherichia coli and Staphylococcus aureus treated with sub-MICs of imipenem

Enhanced phagocytosis, killing, and serum sensitivity of Escherichia coli and Staphylococcus aureus treated with sub-MICs of imipenem

Effect of Butylated Hydroxyanisole and Some of Its Derivatives on Human Neutrophil Oxidative Burst: Chemiluminescence Evaluation

Influence of Sub-Minimum Inhibitory Concentrations of Cefodizime on the Phagocytosis, Intracellular Killing and Oxidative Bursts of Human Polymorphonuclear Leukocytes

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