The effects of serum components on the agglutination of Gram-negative bacteria

1970 ◽  
Vol 16 (2) ◽  
pp. 121-124
Author(s):  
L. Melching ◽  
S. I. Vas
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Enzymatic Reaction ◽  
Bactericidal Effect ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Fresh Serum ◽  
Agglutination Titer ◽  
Plate Counts ◽  
The Complement System ◽  
Serum Components

Fresh normal guinea pig serum was found to increase the agglutination titer of antiserum. Absorption of fresh serum with homologous bacterial cells did not remove the activity. Exposure of fresh serum to 56 C for 30 min, 0.01 M ethylenediaminetetraacetic acid (EDTA), or 150 μg/ml of lipopolysaccharide significantly decreased its activity. This reaction was shown to be initiated at 37 C, but not at 0 C, thus indicating an enzymatic reaction, possibly that of the complement system. Other sources of fresh serum were also found to mediate this reaction. Although this phenomenon requires further investigation, it should be considered when evaluating the bactericidal effect of serum from plate counts, as some decrease in numbers could be due to agglutination.

Bacterial cell damage by the complement system of channel catfish, Ictalurus punctatus

1987 ◽  
Vol 45 ◽  
pp. 888-889
Author(s):  
Rosemarie Rosell-Davis ◽  
Jill A. Jenkins ◽  
Lewis B. Coons ◽  
Donald D. Ourth
Keyword(s):  
Ictalurus Punctatus ◽  
Complement System ◽  
Channel Catfish ◽  
Bacterial Cell ◽  
Cell Damage ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
The Complement System

The alternative complement pathway (ACP) provides the non-immune channel catfish with protection against many Gram-negative bacteria. The role of serum complement against Gram-negative bacteria is death of cells by insertion of the membrane attack complex (C5b-9) into the cell membrane. The assembly of the membrane attack complex is generated by the ACP and is activated by bacterial cell wall components. Pseudomonas fluorescens is a pathogen of channel catfish. In this study, bacteria were examined after incubation with catfish serum by scanning and transmission electron microscopy (SEM and TEM) for ultrastructural evidence of cell envelope damage by the complement system.A percent bactericidal assay determined that catfish plasma was 99% bactericidal against a 24 h culture of P. fluorescens (ATCC 13525). Following a 1 h incubation at 30°C of bacterial dilutions with equal volumes of serum, heat-inactivated serum, zymosan-adsorbed serum, or saline, the bacterial cells were filtered onto 0.22 um nuclepore filters, fixed in glutaraldehyde, dehydrated in ethanol, critical point dried, sputter coated with 15 nm gold and imaged using a JEOL SEM.

Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilisation envelopes of rainbow trout eggs

Zygote ◽  
2000 ◽  
Vol 8 (3) ◽  
pp. 257-265 ◽  
Author(s):  
Shigeharu Kudo
Keyword(s):  
Rainbow Trout ◽  
Ultrastructural Level ◽  
Bactericidal Effect ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Positive ◽  
Bacterial Dna ◽  
Different Types ◽  
Subsequent Degradation ◽  
Vitelline Envelope

Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.

Effect of piperacillin on morphology and viability of gram-negative bacteria

1984 ◽  
Vol 42 ◽  
pp. 218-219
Author(s):  
R. H. Liss
Keyword(s):  
Inhibitory Concentration ◽  
Cytoplasmic Membrane ◽  
Drug Binding ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Drug Induced ◽  
Penicillin Binding Proteins ◽  
Lactam Antibiotic ◽  
Drug Free ◽  
Tube Dilution

Piperacillip (PIP) is b-[D(-)-α-(4-ethy1-2,3-dioxo-l-piperzinylcar-bonylamino)-α-phenylacetamido]-penicillanate. The broad spectrum semisynthetic β-lactam antibiotic is believed to effect bactericidal activity through its affinity for penicillin-binding proteins (PBPs), enzymes on the bacterial cytoplasmic membrane that control elongation and septation during cell growth and division. The purpose of this study was to correlate penetration and binding of 14C-PIP in bacterial cells with drug-induced lethal changes assessed by microscopic, microbiologic and biochemical methods.The bacteria used were clinical isolates of Escherichia coli and Pseudomonas aeruginosa (Figure 1). Sensitivity to the drug was determined by serial tube dilution in Trypticase Soy Broth (BBL) at an inoculum of 104 organisms/ml; the minimum inhibitory concentration of piperacillin for both bacteria was 1 μg/ml. To assess drug binding to PBPs, the bacteria were incubated with 14C-PIP (5 μg/0.09 μCi/ml); controls, in drug-free medium.

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.

Sausage Casings as a Model for Attachment of Salmonella to Meat

1993 ◽  
Vol 56 (5) ◽  
pp. 390-394 ◽  
Author(s):  
ISABEL WALLS ◽  
PETER H. COOKE ◽  
ROBERT C. BENEDICT ◽  
ROBERT L. BUCHANAN
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Bacterial Attachment ◽  
Bacterial Cells ◽  
Inoculum Level ◽  
Plate Counts ◽  
Increasing Temperature ◽  
Convenient Model ◽  
Sausage Casing ◽  
Scanning Electron

Artificial sausage casings were used as a model for studying bacterial attachment to meat connective tissue. Sausage casings of known mass were exposed to suspensions of Salmonella typhimurium in 0.15 M NaCl under various time, temperature, and inoculum level regimes, then washed to remove unattached bacteria. Attached bacterial cells were enumerated using both plate counts and scanning electron microscopy. Bacterial cells attached to sausage casing surfaces within 1 min of incubation. Numbers of attached cells increased with increasing temperature and inoculum levels and with time. Rates of attachment of S. typhimurium to sausage casings were comparable with those reported for attachment to meat surfaces. Sausage casings appear to be a convenient model for examining mechanisms of bacterial attachment to meats.

scholarly journals The kfrA gene is the first in a tricistronic operon required for survival of IncP-1 plasmid R751

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1621-1637 ◽  
Author(s):  
Malgorzata Adamczyk ◽  
Patrycja Dolowy ◽  
Michal Jonczyk ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Coiled Coil ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
C Terminus ◽  
Low Copy Number ◽  
And Control ◽  
Broad Host Range Plasmids

The kfrA gene of the IncP-1 broad-host-range plasmids is the best-studied member of a growing gene family that shows strong linkage to the minimal replicon of many low-copy-number plasmids. KfrA is a DNA binding protein with a long, alpha-helical, coiled-coil tail. Studying IncP-1β plasmid R751, evidence is presented that kfrA and its downstream genes upf54.8 and upf54.4 were organized in a tricistronic operon (renamed here kfrA kfrB kfrC), expressed from autoregulated kfrAp, that was also repressed by KorA and KorB. KfrA, KfrB and KfrC interacted and may have formed a multi-protein complex. Inactivation of either kfrA or kfrB in R751 resulted in long-term accumulation of plasmid-negative bacteria, whereas wild-type R751 itself persisted without selection. Immunofluorescence studies showed that KfrAR751 formed plasmid-associated foci, and deletion of the C terminus of KfrA caused plasmid R751ΔC 2 kfrA foci to disperse and mislocalize. Thus, the KfrABC complex may be an important component in the organization and control of the plasmid clusters that seem to form the segregating unit in bacterial cells. The studied operon is therefore part of the set of functions needed for R751 to function as an efficient vehicle for maintenance and spread of genes in Gram-negative bacteria.

scholarly journals Human genetic deficiencies reveal the roles of complement in the inflammatory network: Lessons from nature

2009 ◽  
Vol 106 (37) ◽  
pp. 15861-15866 ◽  
Author(s):  
Knut Tore Lappegård ◽  
Dorte Christiansen ◽  
Anne Pharo ◽  
Ebbe Billmann Thorgersen ◽  
Bernt Christian Hellerud ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Gram Negative Bacteria ◽  
Enzyme Release ◽  
Experimental Conditions ◽  
Gram Negative ◽  
Cytokines And Chemokines ◽  
Ifn Γ ◽  
Inducible Protein ◽  
The Complement System

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies—nature's own knockouts—including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1β and IL-8 were more dependent on complement than IFN-γ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-γ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria.

scholarly journals A Novel Weissella cibaria Strain UTNGt21O Isolated from Wild Solanum quitoense Fruit: Genome Sequence and Characterization of a Peptide with Highly Inhibitory Potential toward Gram-Negative Bacteria

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1242
Author(s):  
Gabriela N. Tenea ◽  
Pamela Hurtado ◽  
Clara Ortega
Keyword(s):  
Cell Death ◽  
Ethylenediaminetetraacetic Acid ◽  
Sequence Similarity ◽  
Genome Mining ◽  
Chelating Agent ◽  
Target Cells ◽  
Bacterial Cells ◽  
Sequencing Data ◽  
E Coli ◽  
Weissella Cibaria

A novel Weissella cibaria strain UTNGt21O from the fruit of the Solanum quitoense (naranjilla) shrub produces a peptide that inhibits the growth of both Salmonella enterica subsp. enterica ATCC51741 and Escherichia coli ATCC25922 at different stages. A total of 31 contigs were assembled, with a total length of 1,924,087 bases, 20 contig hits match the core genome of different groups within Weissella, while for 11 contigs no match was found in the database. The GT content was 39.53% and the genome repeats sequences constitute around 186,760 bases of the assembly. The UTNGt21O matches the W. cibaria genome with 83% identity and no gaps (0). The sequencing data were deposited in the NCBI Database (BioProject accessions: PRJNA639289). The antibacterial activity and interaction mechanism of the peptide UTNGt21O on target bacteria were investigated by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with different concentrations (1×, 1.5× and 2× MIC) of the peptide applied alone or in combination with chelating agent ethylenediaminetetraacetic acid (EDTA) at 20 mM. The results indicated a bacteriolytic effect at both early and late target growth at 3 h of incubation and total cell death at 6 h when EDTA was co-inoculated with the peptide. Based on BAGEL 4 (Bacteriocin Genome Mining Tool) a putative bacteriocin having 33.4% sequence similarity to enterolysin A was detected within the contig 12. The interaction between the peptide UTNGt21O and the target strains caused permeability in a dose-, time- response manner, with Salmonella (3200 AU/mL) more susceptible than E. coli (6400 AU/mL). The results indicated that UTNGt21O may damage the integrity of the cell target, leading to release of cytoplasmic components followed by cell death. Differences in membrane shape changes in target cells treated with different doses of peptide were observed by transmission electronic microscopy (TEM). Spheroplasts with spherical shapes were detected in Salmonella while larger shaped spheroplasts with thicker and deformed membranes along with filamentous cells were observed in E. coli upon the treatment with the UTNGt21O peptide. These results indicate the promising potential of the putative bacteriocin released by the novel W. cibaria strain UTNGt21O to be further tested as a new antimicrobial substance.

scholarly journals Elimination of Extracellular Adenosine Triphosphate for the Rapid Prediction of Quantitative Plate Counts in 24 h Time-Kill Studies against Carbapenem-Resistant Gram-Negative Bacteria

2020 ◽  
Vol 8 (10) ◽  
pp. 1489
Author(s):  
Yiying Cai ◽  
Jonathan J. Ng ◽  
Hui Leck ◽  
Jocelyn Q. Teo ◽  
Jia-Xuan Goh ◽  
...  
Keyword(s):  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Infection Models ◽  
Rapid Prediction ◽  
Atp Bioluminescence ◽  
Plate Counts ◽  
Time Kill

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.

Sign in / Sign up

Export Citation Format

Share Document