scholarly journals Rapid and Reliable Detection of Antimicrobial Peptide Penetration into Gram-Negative Bacteria Based on Fluorescence Quenching

2009 ◽  
Vol 53 (8) ◽  
pp. 3501-3504 ◽  
Author(s):  
Monica Benincasa ◽  
Sabrina Pacor ◽  
Renato Gennaro ◽  
Marco Scocchi
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Quenching ◽  
Polymyxin B ◽  
Mechanisms Of Action ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Cell Penetration ◽  
Gram Negative ◽  
Reliable Detection ◽  
Rapid Flow

ABSTRACT In this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are internalized into bacterial cells and differentiate them from those that are membrane active. The method was applied to fluorescently labeled Bac71-35 and polymyxin B, whose mechanisms of action are, respectively, based on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former mechanism is of considerable interest for the intracellular delivery of membrane-impermeant drugs.

scholarly journals Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry

2010 ◽  
Vol 76 (15) ◽  
pp. 5088-5096 ◽  
Author(s):  
Mohiuddin M. Taimur Khan ◽  
Barry H. Pyle ◽  
Anne K. Camper
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Syringae ◽  
Phase 1 ◽  
Epifluorescence Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Gram Negative ◽  
Analytical Technique

ABSTRACT An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

scholarly journals Polymyxin B Induces Lysis of Marine Pseudoalteromonads

2007 ◽  
Vol 51 (11) ◽  
pp. 3908-3914 ◽  
Author(s):  
Mart Krupovič ◽  
Rimantas Daugelavičius ◽  
Dennis H. Bamford
Keyword(s):  
Divalent Cations ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Cytoplasmic Membranes ◽  
Long Time ◽  
High Concentrations ◽  
Cell Envelopes

ABSTRACT Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that “what is true for Escherichia coli is true for all bacteria” does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.

scholarly journals LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Tian ◽  
Guillaume Manat ◽  
Elise Gasiorowski ◽  
Rodolphe Auger ◽  
Samia Hicham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Lipid A ◽  
Negative Charge ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

Production and use of monoclonal antibodies for identification of Bradyrhizobium japonicum strains

1988 ◽  
Vol 34 (1) ◽  
pp. 88-92 ◽  
Author(s):  
D. Velez ◽  
J. D. Macmillan ◽  
L. Miller
Keyword(s):  
Monoclonal Antibodies ◽  
Bradyrhizobium Japonicum ◽  
Chemical Nature ◽  
Periodate Oxidation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Polyclonal Antisera ◽  
Somatic Antigens

Thirteen murine hybridomas capable of producing monoclonal antibodies to somatic antigens on Bradyrhizobium japonicum were developed and an indirect enzyme-linked immunosorbent assay was used to test reactivity of the antibodies against 20 strains of B. japonicum. Although polyclonal antisera from mice immunized with strains of B. japonicum reacted with bacterial cells of all 20 strains, individual monoclonals were more specific. Some antibodies reacted with as few as 2 and one with as many as 11 strains. On the basis of reactivity with the set of 13 monoclonal antibodies, the 20 strains of B. japonicum could be divided arbitrarily into five groups. Three of five monoclonal antibodies tested reacted with bacteroids taken directly from soybean nodules. One monoclonal bound to cells of five species of Rhizobium, but none of the 13 reacted with gram-negative bacteria representing six other genera. Treatment of cells with reagents and heat indicated the chemical nature of the antigens to five of the monoclonals. Antigen reactive with one antibody was destroyed by periodate oxidation indicating that it was a polysaccharide. Two antigens were probably proteins as they could be digested by trypsin and denatured by heat. Two others were inactivated by all three treatments suggesting they were glycoproteins.

scholarly journals Polymyxin B Nephrotoxicity and Efficacy against Nosocomial Infections Caused by Multiresistant Gram-Negative Bacteria

2003 ◽  
Vol 47 (8) ◽  
pp. 2659-2662 ◽  
Author(s):  
John P. Ouderkirk ◽  
Jill A. Nord ◽  
Glenn S. Turett ◽  
Jay Ward Kislak
Keyword(s):  
Renal Failure ◽  
Renal Function ◽  
Renal Toxicity ◽  
Clinical Information ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Length Of Treatment ◽  
Baseline Renal Function ◽  
Overall Mortality

ABSTRACT Reported rates of nephrotoxicity associated with the systemic use of polymyxins have varied widely. The emergence of infections due to multiresistant gram-negative bacteria has necessitated the use of systemic polymyxin B once again for the treatment of such infections. We retrospectively investigated the rate of nephrotoxicity in patients receiving polymyxin B parenterally for the treatment of infections caused by multiresistant gram-negative bacteria from October 1999 to September 2000. Demographic and clinical information was obtained for 60 patients. Outcome measures of interest were renal toxicity and clinical and microbiologic efficacy. Renal failure developed in 14% of the patients, all of whom had normal baseline renal function. Development of renal failure was independent of the daily and cumulative doses of polymyxin B and the length of treatment but was significantly associated with older age (76 versus 59 years, P = 0.02). The overall mortality was 20%, but it increased to 57% in those who developed renal failure. The organism was cleared in 88% of the patients from whom repeat specimens were obtained. The use of polymyxin B to treat multiresistant gram-negative infections was highly effective and associated with a lower rate of nephrotoxicity than previously described.

scholarly journals Visualizing the Potential Impairment of Polymyxin B to Central Nervous System Through MR Susceptibility-Weighted Imaging

2021 ◽  
Vol 12 ◽  
Author(s):  
Ni Zhang ◽  
Lichong Zhu ◽  
Qiuhong Ouyang ◽  
Saisai Yue ◽  
Yichun Huang ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Polymyxin B ◽  
Susceptibility Weighted Imaging ◽  
Gram Negative Bacteria ◽  
Severe Toxicity ◽  
Gram Negative ◽  
The Impact

Polymyxin B (PMB) exert bactericidal effects on the cell wall of Gram-negative bacteria, leading to changes in the permeability of the cytoplasmic membrane and resulting in cell death, which is sensitive to the multi-resistant Gram-negative bacteria. However, the severe toxicity and adverse side effects largely hamper the clinical application of PMB. Although the molecular pathology of PMB neurotoxicity has been adequately studied at the cellular and molecular level. However, the impact of PMB on the physiological states of central nervous system in vivo may be quite different from that in vitro, which need to be further studied. Therefore, in the current study, the biocompatible ultra-uniform Fe3O4 nanoparticles were employed for noninvasively in vivo visualizing the potential impairment of PMB to the central nervous system. Systematic studies clearly reveal that the prepared Fe3O4 nanoparticles can serve as an appropriate magnetic resonance contrast agent with high transverse relaxivity and outstanding biosafety, which thus enables the following in vivo susceptibility-weighted imaging (SWI) studies on the PMB-treated mice models. As a result, it is first found that the blood-brain barrier (BBB) of mice may be impaired by successive PMB administration, displaying by the discrete punctate SWI signals distributed asymmetrically across brain regions in brain parenchyma. This result may pave a noninvasive approach for in-depth studies of PMB medication strategy, monitoring the BBB changes during PMB treatment, and even assessing the risk after PMB successive medication in multidrug-resistant Gram-negative bacterial infected patients from the perspective of medical imaging.

Polymyxin B-modified conjugated oligomer nanoparticle for targeted identification and enhanced photodynamic antimicrobial therapy

2021 ◽  
Author(s):  
Qi Jiang ◽  
Xinrong Yan ◽  
Dan Jiao ◽  
Jiangyan Zhang ◽  
Yonggang Wu ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Antimicrobial Therapy ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Photodynamic Antimicrobial Therapy

We report a photosensitive polymyxin B-modified conjugated oligomer nanoparticle that integrates the targeted identification and synergistic photodynamic therapy in one treatment against resistant gram-negative bacteria. The study expands the application...

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