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Detection of invasiveness of mammalian cells by Escherichia coli: collaborative study
Abstract:
In a collaborative study of 2 standard procedures (Procedure I, presented in J. Assoc. Off. Anal. Chem. 60, 546-562 (1977) and Procedure II in the Bacteriological Analytical Manual, 1978), 7 laboratories evaluated the invasive potential of Escherichia coli. Monolayers of HeLa cultures were infected with E. coli (infectivity ratio 100 bacteria/mammalian cell) suspended in 2 menstrua: heat-inactivated fetal bovine serum (Procedure I) and 0.2% bovine albumin Fraction V dissolved in Earle's buffered salts (Procedure II). After uptake of the bacteria, intracellular growth, and differential staining, the percentages of HeLa cells containing a minimum of 5 bacteria were determined microscopically. All laboratories correctly identified invasiveness of E. coli by Procedure I; 5 of 6 laboratories recognized invasiveness by Procedure II. Although Procedure I was more reproducible than Procedure II, repeatability did not differ significantly. Procedure I was adopted official first action for presumptive recognition of invasiveness in E. coli.
Insights
Procedure I accurately identified the invasive potential of Escherichia coli in a collaborative study. This method was adopted for presumptive recognition of invasiveness, showing better reproducibility than Procedure II.
Area of Science:
- Microbiology
- Cell Biology
Background:
- Assessing bacterial invasiveness is crucial for understanding pathogenesis.
- Standardized methods are needed for reliable evaluation of bacterial invasion potential.
Purpose of the Study:
- To collaboratively evaluate two standard procedures for assessing the invasive potential of Escherichia coli.
- To determine the reproducibility and accuracy of each procedure in identifying bacterial invasiveness.
Main Methods:
- Seven laboratories infected HeLa cell monolayers with Escherichia coli using two different suspending media (menstrua).
- Procedure I utilized heat-inactivated fetal bovine serum, while Procedure II used bovine albumin dissolved in Earle's buffered salts.
- Microscopic examination quantified the percentage of HeLa cells containing at least 5 bacteria post-infection.
Main Results:
- Procedure I correctly identified the invasiveness of E. coli in all participating laboratories.
- Procedure II correctly identified invasiveness in 5 out of 6 laboratories.
- Procedure I demonstrated superior reproducibility compared to Procedure II, though repeatability was not significantly different.
Conclusions:
- Procedure I is a reliable and reproducible method for the presumptive recognition of invasiveness in E. coli.
- The study led to the adoption of Procedure I as an official first action method.
- Differences in menstrua significantly impact the evaluation of bacterial invasiveness.