Determination of serum bactericidal activity against Escherichia coli by an automated photometric method

Abstract

The resistance of gram-negative bacteria to complement-mediated serum activity is supposedly an important virulence factor. However, the lack of standardization in the methods used to determine serum activity and the many definitions applied make the comparisons between studies very difficult. We developed a rapid photometric method that we compared with a classical killing one. Escherichia coli in the exponential phase of growth in brain heart infusion broth (final inoculum, 107 CFU/ml) at 35°C was added to 50% human serum in Veronal buffer. Viable counts and automatic recording of the variations in the optical densities were obtained for 40 E. coli strains isolated from the stools of healthy adults. With the viable count method, 17 (42.5%) were susceptible (at least a 1 log CFU/ml decrease), 17 (42.5%) were resistant (a 0.6 log CFU/ml increase), 4 (10%) were intermediate (poorly growing inoculum or a decrease of less than 1 log CFU/ml), and 2 could not be classified (nonreproducible results). Agreement between both methods was observed for 87.5% of the stool strains. Eight reference strains of known susceptibilities were classified identically by both methods, leading to a final concordance rate of 89.6%. A total of 129 blood isolates were tested by the photometric method: 64 (49.6%) were resistant, 50 (38.8%) were susceptible 5 (3.9%) showed early regrowth, and 10 (7.7%) were not perfectly reproducible. Of these 129 blood isolates, s were also tested by the killing method: 37 (49%) were resistant, 32 (43%) were susceptible, and 6 (8%) were intermediate. The concordance rate between both assays was 89% for the blood isolates; when the minor discordances were ruled out, it was 97%. This automated method could be a useful screening tool for detecting resistance to serum in clinical trials and for studying the in vitro variations of this property.

Effects of fatty acids and calf starter form on intake, growth, digestion, and selected blood metabolites in male calves from 0 to 4 months of age

“…Blood was collected at approximately 1000 h each d into evacuated tubes without anticoagulant and allowed to clot for approximately 1 h. Serum was separated by centrifugation at 3,000 × g for 20 min at room temperature and placed in labeled tubes and frozen at −20°C until shipped to the laboratory for analysis of haptoglobin, serum bactericidal activity (SBA), and cortisol. Serum bactericidal activity against a live culture of bacteria was measured using methods similar to bactericidal activity of plasma (Crokaert et al, 1988;Moisá et al, 2018). Serum was incubated with Escherichia coli 8739 at a 1:8 ratio for 10 min.…”

Virulence of a T6SS Campylobacter jejuni chicken isolate from North Romania

“…Therefore, the higher adhesion and invasion levels observed here could be attributed to the fact that SV12 harbours a functional T6SS system. C. jejuni must be able to withstand the bile salts secreted into the intestinal tract and the bactericidal effects of the human and animal serum in order to cause infection [17]. In the present study, although both strains were susceptible to bile salts, SV12 showed increased survival compared to the C. jejuni 81–176 control strain.…”

Effects of tissue-specific biomolecules on piglets after-weaning period

“…To study the animal resistance on 28 th day of the experiment, the integral index of BSAA was determined using a culture of E. coli. E. coli (24 h culture) preparation, which consists of a dilution to 2×10 9 CFU in 1 mL of sterile saline solution (ZOMZ, Russia), followed by addition of 10 μL of the microbial suspension obtained to 4.5 mL of meat-peptone broth and incubation for 24 h (daily culture broth) [20].…”