Antimicrobial action of rabbit leukocyte CAP18(106-137).

1997 ◽  
Vol 41 (3) ◽  
pp. 624-629 ◽  
Author(s):  
D J Mason ◽  
R Dybowski ◽  
J W Larrick ◽  
V A Gant
Keyword(s):  
Membrane Potential ◽  
Colony Formation ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Broth Culture ◽  
Antimicrobial Protein ◽  
Culture Techniques ◽  
E Coli ◽  
Conventional Culture ◽  
Solid Media

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.

scholarly journals Evaluation of Physiological Effects Induced by Manuka Honey Upon Staphylococcus aureus and Escherichia coli

2019 ◽  
Vol 7 (8) ◽  
pp. 258 ◽  
Author(s):  
Patricia Combarros-Fuertes ◽  
Leticia M. Estevinho ◽  
Rita Teixeira-Santos ◽  
Acácio G. Rodrigues ◽  
Cidália Pina-Vaz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Membrane Potential ◽  
Membrane Integrity ◽  
Efflux Pumps ◽  
Coli Strain ◽  
Antibacterial Action ◽  
Manuka Honey ◽  
E Coli ◽  
Action Mechanisms

Several studies have explored the antimicrobial properties of manuka honey (MkH). However, the data available regarding antibacterial action mechanisms are scarcer. The aim of this study was to scrutinize and characterize primary effects of manuka honey (MkH) upon the physiological status of Staphylococcus aureus and Escherichia coli (as Gram-positive and Gram-negative bacteria models, respectively), using flow cytometry (FC) to reveal its antibacterial action mechanisms. Effects of MkH on membrane potential, membrane integrity and metabolic activity were assessed using different fluorochromes in a 180 min time course assay. Time-kill experiments were carried out under the same conditions. Additionally, MkH effect on efflux pumps was also studied in an E. coli strain with an over-expression of several efflux pumps. Exposure of bacteria to MkH resulted in physiological changes related to membrane potential and membrane integrity; these effects displayed slight differences among bacteria. MkH induced a remarkable metabolic disruption as primary physiological effect upon S. aureus and was able to block efflux pump activity in a dose-dependent fashion in the E. coli strain.

scholarly journals Occurrence of Escherichia coli in cloacal samples of broiler chicken from Kollam and Kottayam districts

2021 ◽  
Vol 52 (4) ◽  
Author(s):  
Afsal S. ◽  
Latha C. ◽  
Sethulekshmi C. ◽  
Binsy M. ◽  
Beena C. J. ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Broiler Chicken ◽  
One Health ◽  
Holistic Approach ◽  
Cloacal Swab ◽  
Zoonotic Diseases ◽  
Foodborne Illness ◽  
Culture Techniques ◽  
E Coli ◽  
Conventional Culture

Foodborne pathogens like E. coli are considered as the major causes of foodborne illness in humans worldwide. The present study was undertaken to determine the occurrence of E. coli in cloacal samples of broiler chicken from Kollam and Kottayam districts. The occurrence of E. coli in cloacal samples from broiler chicken was 76.5 per cent from Kollam and 79 per cent from Kottayam through culture techniques. Out of the total 400 cloacal swab samples collected from broiler chicken, 77.8 per cent were positive for E. coli. The samples which were subjected to conventional culture techniques were further analysed for PCR confirmation. The study revealed that, 56.5 and 67 per cent samples were positive for E. coli from Kollam and Kottayam, respectively. An overall occurrence of 61.8 per cent out of 400 samples were confirmed for E. coli by PCR. One Health approach can be used as a suitable tool to combat the foodborne zoonotic diseases, since it is an integrated, multidisciplinary, holistic approach. Proper implementation of biosecurity measures in farms is mandatory to control foodborne zoonotic diseases.

scholarly journals Biofilms facilitate cheating and social exploitation of β-lactam resistance in Escherichia coli

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Elli Amanatidou ◽  
Andrew C. Matthews ◽  
Ute Kuhlicke ◽  
Thomas R. Neu ◽  
James P. McEvoy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Social Interactions ◽  
Social Protection ◽  
Conjugative Plasmid ◽  
Broth Culture ◽  
Bacterial Strains ◽  
Gram Negative ◽  
E Coli ◽  
Solid Media ◽  
High Doses

AbstractGram-negative bacteria such as Escherichia coli commonly resist β-lactam antibiotics using plasmid-encoded β-lactamase enzymes. Bacterial strains that express β-lactamases have been found to detoxify liquid cultures and thus to protect genetically susceptible strains, constituting a clear laboratory example of social protection. These results are not necessarily general; on solid media, for instance, the rapid bactericidal action of β-lactams largely prevents social protection. Here, we tested the hypothesis that the greater tolerance of biofilm bacteria for β-lactams would facilitate social interactions. We used a recently isolated E. coli strain, capable of strong biofilm formation, to compare how cooperation and exploitation in colony biofilms and broth culture drives the dynamics of a non-conjugative plasmid encoding a clinically important β-lactamase. Susceptible cells in biofilms were tolerant of ampicillin—high doses and several days of exposure were required to kill them. In support of our hypothesis, we found robust social protection of susceptible E. coli in biofilms, despite fine-scale physical separation of resistant and susceptible cells and lower rates of production of extracellular β-lactamase. In contrast, social interactions in broth were restricted to a relatively narrow range of ampicillin doses. Our results show that β-lactam selection pressure on Gram-negative biofilms leads to cooperative resistance characterized by a low equilibrium frequency of resistance plasmids, sufficient to protect all cells.

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Life without Division: Physiology ofEscherichia coliFtsZ-Deprived Filaments

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Alicia Sánchez-Gorostiaga ◽  
Pilar Palacios ◽  
Rocío Martínez-Arteaga ◽  
Manuel Sánchez ◽  
Mercedes Casanova ◽  
...  
Keyword(s):  
Solid Medium ◽  
Membrane Integrity ◽  
Test Tube ◽  
Antimicrobial Compounds ◽  
Liquid Form ◽  
Altered Expression ◽  
E Coli ◽  
Ftsz Ring ◽  
Altered Expression Pattern ◽  
The Stability

ABSTRACTWhen deprived of FtsZ,Escherichia colicells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.IMPORTANCEOur results suggest a role for FtsZ, in addition to its already known effect in the constriction ofE. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated byE. coli, an observation that may help in the design of effective antimicrobial compounds.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus andMicrococcus luteus

2000 ◽  
Vol 44 (4) ◽  
pp. 827-834 ◽  
Author(s):  
David J. Novo ◽  
Nancy G. Perlmutter ◽  
Richard H. Hunt ◽  
Howard M. Shapiro
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Membrane Permeability ◽  
Micrococcus Luteus ◽  
Flow Cytometric Analysis ◽  
Bacterial Counts ◽  
Flow Cytometric ◽  
Permeability Parameter ◽  
Particle Counts

ABSTRACT Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus andMicrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

scholarly journals Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection

2016 ◽  
Vol 2016 ◽  
pp. 1-6
Author(s):  
Michael J. Jarrett ◽  
Andres Vázquez-Torres ◽  
Daniel N. Frank ◽  
Bruce D. McCollister ◽  
Patrick K. Henthorn ◽  
...  
Keyword(s):  
Colony Formation ◽  
Rrna Gene ◽  
Contributing Factors ◽  
Vaginal Cuff ◽  
Pelvic Infection ◽  
Bacterial Colony ◽  
E Coli ◽  
Tissue Sealant

Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff.Methods.Escherichia coliwas incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015.In vitrotechniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff.Results. Mean bacterial colony formation in PBS was greater forE. coliincubated in the presence of GTM (1.48 × 107 CFU/mL) versus without (9.95 × 105 CFU/mL) following 20-hour incubation (p=0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) withEnterococcus faecalis.Conclusion.In vitroexperiments support a facilitating role of GTM on colony formation ofE. coliin PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence ofE. faecalis, but results were inconclusive. Further studies are recommended.

scholarly journals IN VITRO MICROBIAL TIME-KILLING CURVE FOR NEWLY SYNTHESIZED AMINOACETYLENIC-2-MERCAPTOBENZOTHIAZOLE COMPOUND

2017 ◽  
Vol 9 (10) ◽  
pp. 125
Author(s):  
Aseel Alsarahni ◽  
Zuhair Muhi Eldeen ◽  
Elham Al-kaissi ◽  
Hiba Al-malliti
Keyword(s):  
Minimum Bactericidal Concentration ◽  
Viable Count ◽  
Broth Culture ◽  
Dilution Method ◽  
Time Intervals ◽  
Minimum Fungicidal Concentration ◽  
E Coli ◽  
Different Types ◽  
Broth Dilution

Objective: To determine the time needed for killing different types of microorganisms by a newly synthesized 2-mercapto-1,3-benzothiazole derivative in comparison to ciprofloxacin and fluconazole.Methods: The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) for 2-{[4-(2,6-dimethylPiperidin-1-yl)but-2-yn-1-yl]Sulfanyl}-1,3-benzothiazole(AZ3) compound were determined, using the broth dilution method. The MBC and MFC dilutions were prepared. Broth cultures of Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) were incubated at 37 °C for 24 h, and Candida albicans (C. albicans) was incubated at 25 °C for 48 h. 0.1 ml of each broth culture represent 1.5 x 106 CFU/ml was challenged with 9.9 ml broth containing the MBC or MFC concentrations of the AZ3 compound. From each sample at different time intervals, 1 ml was taken and added to 9 ml of sterile distilled water, in order to neutralize the effect of AZ3. Serial dilution was done and a viable count was determined from the appropriate dilutions.Results: The viability of the P. aeruginosa, E. coli, S. aureus, B. subtilis and C. albicans were killed within 3.5 h, 5 h, 24 h, 3 h and 5 h respectively. The time killing curves showed that AZ3 needed longer time for killing S. aureus than the time needed to kill B. subtilis. On the other hand, AZ3 needed a shorter time to kill P. aeruginosa, than the time needed to kill E. coli. In comparison with ciprofloxacin, AZ3 needed a shorter time to kill P. aeruginosa and E. coli, and the same time to kill B. subtilis, while it needed longer time than ciprofloxacin to kill S. aureus. In comparison with fluconazole, AZ3 with lower MFC than fluconazole needed longer time to kill C. albicans.Conclusion: AZ3 showed promising antimicrobial killing activities, in compared with ciprofloxacin and fluconazole, which promoted our interest to investigate the time of killing needed for other 2-mercaptobenzothiazole derivatives against different types of microorganisms.

scholarly journals The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

2005 ◽  
Vol 187 (2) ◽  
pp. 729-738 ◽  
Author(s):  
Elizabeth A. Marcus ◽  
Amiel P. Moshfegh ◽  
George Sachs ◽  
David R. Scott
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Potential ◽  
Carbonic Anhydrase ◽  
Membrane Integrity ◽  
Carbonic Anhydrase Activity ◽  
Knockout Mutant ◽  
Inner Membrane ◽  
Acid Acclimation

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

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