Methodology for Recognition of Invasive Potential of Escherichia coli

Publisher Website
Google Scholar
Abstract
Surveillance for dysentery-related invasive potential in bacteria using the Sereny keratoconjunctivitis test is restricted by expense, time factor, and necessity for confirmation. Primary screening of isolates in a standardized mammalian cell culture system is recommended. Bacteria are grown 20 hr in veal infusion, washed, and resuspended in 20% heat-inactivated fetal bovine serum (FBS) supplemented with 0.12% brain heart infusion and 0.1% bile salts. The HeLa culture is grown 20 hr as a monolayer in chamber slides with 90% minimal essential medium (MEM)-10% FBS. The host culture is infected at a ratio of 10 bacteria/ mammalian cell for 3 hr at 35°C. The infection medium is replaced with MEM-FBS supplemented with 300 μg lysozyme and 5 μg gentamycin/ml. The infected monolayer is incubated 5 hr at 35°C to permit intracellular multiplication. Specimens arc washed, fixed with methanol, and stained successively with May-Grunwald and Giemsa dyes. Bacteria occur within the cytoplasm if invasion has occurred. The criterion for a positive test is that 1% of the host cells possesses at least 5 bacteria in 2 of 3 trials. Invasiveness is correlated with and possibly pre-conditioned by cytotoxic principle(s). Infectivity rates vary from 0 to 30%. The cytopathic effect is noted in 5–50% of HeLa cells. Positive results must be confirmed by the Sereny test.