A rapid turbidimetric assay for the study of serum sensitivity of Escherichia coli

Pelkonen, Sinikka

doi:10.1016/0378-1097(87)90497-6

Cited by 11 publications

(15 citation statements)

References 6 publications

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“…To prepare 20 ml of gel solution we mixed 10 ml of the acrylamide stock solution, 2 ml of the concentrated buffer, and 7.9 ml of H20 and initiated polymerization by adding 7.5 ,ul of tetramethylethylenediamine and 75 ,ul of 10% ammonium persulfate. Mixtures were poured into glass plates (15-cm wide and 0.8-mm thick) differing in length (14,25, and 37 cm) at room temperature. The longer plates were swept with dimethyldichlorosilane solution (BDH Chemicals Ltd.) to facilitate the detachment of the gel from the plate.…”

Section: Methodsmentioning

confidence: 99%

“…6A) 25 V/cm for 18 h at 4°C. Before being stained with silver, one-half of the gel was fixed with alcian blue in 2% acetic acid (ac), and the other half was fixed with alcian blue in water (w).…”

Section: Methodsmentioning

confidence: 99%

“…t Present address: National Veterinary Institute, P.O. Box 368, SF-00101 Helsinki, Finland. surfaces (17,25,33). Such a method would also facilitate studies of the molecular interactions in which sialic acid is involved.…”

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confidence: 99%

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Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria

1988

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Methods were developed for the polyacrylamide gel electrophoretic analysis of capsular polysaccharides of bacteria with Escherichia coli Kl as a model. Conditions were determined for the rapid and gentle extraction of the Kl polysaccharide by incubation of the bacteria in a volatile buffer and for the subsequent removal of the putative phospholipid moiety attached to the reducing end of the polysaccharide. Detection of the polysaccharides after gel electrophoresis was carried out by fluorography of samples labeled by sodium borotritiide reduction or by combined alcian blue and silver staining. The smaHest components could be detected only by fluorography, owing to diffusion during staining. Components of the E. coli Kl polysialic acid capsule ranging from monomers to 80 sialic-acid-unit-containing polymers could be separated as distinct bands in a ladderlike pattern. A maximum chain length of 160 to 230 sialyl residues was estimated for the bulk of the Kl polysaccharide from the nearly linear reciprocal relationship between the logarithm of the molecular size and the distance of migration. Gel electrophoresis of capsular polysaccharides of other bacterial species revealed different electrophoretic mobilities for each polysaccharide, with a ladderlike pattern displayed by the fastest-moving components. There are many potential applications of this facile method for the determination of the sizes of molecules present in a polydisperse polysaccharide sample. When combined with the simple method for the isolation of the capsule, as in the case of the Kl capsule, it provides an efficient tool for the characterization and comparison of the capsular polysaccharides of bacteria.Capsular polysaccharides are important bacterial surface determinants that consist of polymers with repeating units of one or more monosaccharides (15,16,30). Characterization of bacterial proteins and lipopolysaccharides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a commonly applied method in comparative and structural studies. Similar methods have not been available for capsular polysaccharides, and their characterization has therefore been hampered by the need for cumbersome analytical procedures. The chain length of the polysaccharides has been determined with methods like gel filtration, ion-exchange chromatography, determination of the ratio of the terminal and internal residues in the polysaccharide chain, and highperformance liquid chromatography (13,19,20,26,30).One of the most extensively studied capsules is the Escherichia coli Kl capsule, a homopolymer with up to 200 sialic acid residues and ax2-8 linkages (4,21,26). Capsular antibodies and capsule-specific bacteriophages seem to recognize conformational determinants in the polysaccharide chain. In the case of the Kl capsule, mono-and polyclonal anti-Kl antibodies (8, 27) require about eight sialyl residues for binding (6, 7), whereas different Kl-specific bacteriophages containing capsule-degrading endosialidases differ in their minimum substrate size and capsule-...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria

1988

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“…The bactericidal effect of human normal serum on enterococcal strains was assessed following the protocol of Pelkonen and Finne [25]. A pool of human normal serum was obtained by heating at 56°C for 30 minutes and adding ethylenglycol 10 mM as well as MgCl 2 5mM in order to inactivate the classical complement pathway.…”

Section: Resistance To Bactericidal Effect Of Normal Serummentioning

confidence: 99%

Clinical and microbiological features of bacteremia caused by Enterococcus faecalis

Ceci

Delpech

Sparo

et al. 2015

J Infect Dev Ctries

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Introduction: Enterococcus faecalis is a frequent etiologic agent of invasive infections in hospitalized patients. The aim of this study was to analyze clinical and microbiological features of bacteremia caused by E. faecalis. Methodology: Between 2011 and 2013, significant bacteremia caused by E. faecalis in hospitalized patients was studied. Patient characteristics, comorbid conditions, and 14-day mortality were recorded. Virulence genes esp, gelE, and cylA; opsonophagocytosis resistance; resistance to bactericidal effect of normal serum; beta lactamase production; and susceptibility to ampicillin, vancomycin, teicoplanin, gentamicin, and streptomycin were investigated. Results: E. faecalis strains were recovered from 33 bacteremic patients. Polymicrobial bacteremia was diagnosed in 2 patients; 10 patients died. Virulence genes were found in strains from both deceased patients and survivors. Sources of bacteremia included urinary tract infections (36.4%), vascular catheters (15.1%), abscesses (9.1%), and unknown (48.5%). Underlying diseases included cancer (30.3%), diabetes (36.4%), cirrhosis (6.1%), renal (36.4%), and chronic obstructive pulmonary disease (2.0%). Co-morbidities included alcohol use (26.1%); glucocorticoid therapy (19.0%); prior antibiotic therapy (60.6%); and central venous (21.2%), arterial (12.1%), and urinary (63.6%) catheters. Also, 57.6% of patients came from the intensive care unit (ICU); 33.3% had mechanical ventilation. Significant mortalityassociated conditions included polymicrobial bacteremia, oncological disease, APACHE II score ≥ 20, ICU stay, renal disease, central venous catheter, and mechanical ventilation. Conclusions: Outcome of patients was associated with their status and not with the presence of virulence genes in E. faecalis strains. A significant percentage of bacteremia had undetermined origin. An alternate origin may be the gastrointestinal tract, through translocation.

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“…The sensitivity of E. faecalis to the bactericidal effect of human normal serum was tested as described by Pelkones and Finne [23]. Bacteria grown in BHI for 18h were diluted in PBS (10 9 bacteria/mL) and 175µL of the bacterial suspension and the same quantity of PBS were pipetted into the wells of microtitre plates, 100µL of serum (final concentration 36%) were added and incubated at 37º C. The absorbance at 630nm was measured at 0, 30, 60, 120 and 180 min.…”

Section: Sensitivity To the Bactericidal Effect Of Human Normal Serummentioning

confidence: 99%

Phenotypic Detection of Virulence Traits and Antibiotic Susceptibility of Endodontic Enterococcus faecalis Isolates

Patidar¹,

Gupta²,

Singh³

2013

AJMR

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Enterococcus faecalis is a Gram-positive member of human gastrointestinal flora that is in recent years emerging as an important cause of endodontic infections. In this study, we have investigated the occurrence of virulence determinants and antibiotic susceptibility pattern of E. faecalis isolates, originating from root canals of apical periodontitis. Among 52 E. faecalis isolates, 32 (61.5%) isolates produced hemolysin on blood agar while all (100%) isolates showed hemolysin production on BHI-GA ((BHI medium supplemented with 1% glucose and 0.03% L-arginine), 18 (34.6%) produced gelatinase, 38 (73%) produced caseinase, no hemagglutination was observed in E. faecalis isolates, whereas all 52 (100%) resistant to serum and formed biofilm. Antibiotic susceptibility results showed that all (100%) E. faecalis isolates were susceptible to amoxicillin, amoxicillin/clavulanate, and vancomycin. Whereas, 32 (61.5%) E. faecalis isolates were resistant to chloramphenicol, 30 (57.6%) isolates were resistant to ciprofloxacin, 39 (75%) isolates were resistant to erythromycin, and 34 (65.3%) isolates were resistant to tetracycline. Multi-drug resistance was observed in 16 (30.7%) isolates of E. faecalis to chloramphenicol, ciprofloxacin, erythromycin and tetracycline antibiotics. These findings demonstrate the presence of putative virulence determinants in E. faecalis isolates originating from root canal and suggest amoxicillin, amoxicillin/clavulanate, and vancomycin as more effective than other antibiotics tested.

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A rapid turbidimetric assay for the study of serum sensitivity of Escherichia coli

Cited by 11 publications

References 6 publications

Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria

Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria

Clinical and microbiological features of bacteremia caused by Enterococcus faecalis

Phenotypic Detection of Virulence Traits and Antibiotic Susceptibility of Endodontic <i>Enterococcus </i><i>f</i><i>aecalis</i> Isolates

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