TABLE 5.

Mutation in barA or uvrY reduces attachment and invasion of E. coli O78:K80:H9 strain χ7122 to chicken embryo fibroblasts

Relevant genotype	No. of cells or bacteria (log10 CFU/ml)a				Attachment indexf	Invasion indexg
	Initial cellsb	Attached and invaded bacteria after 2 hc	Invaded bacterial fraction surviving after 4 hd	Calculated attached bacteriae
Wild type	8.6 ± 0.9	7.1 ± 0.9	6.4 ± 0.8	7.1 ± 0.9	2.7 × 10−2	2.0 × 10−1
barA	8.6 ± 0.9	6.7 ± 0.8	4.8 ± 0.7	6.6 ± 0.8	1.2 × 10−2	1.5 × 10−2
uvrY	8.6 ± 0.9	6.3 ± 0.8	4.0 ± 0.6	6.3 ± 0.8	5.0 × 10−3	5.0 × 10−3
uvrY/p-uvrY	8.6 ± 0.9	7.2 ± 0.9	6.3 ± 0.8	7.1 ± 0.8	4.5 × 10−2	1.3 × 10−2

^aResults represent mean values ± standard deviations from three independent experiments.

^bDF-1 chicken embryo fibroblasts were obtained from the laboratory of S. K. Samal (Virginia-Maryland Regional College of Veterinary Medicine, College Park, Md.). The cells were grown in Dulbecco's modified Eagle's medium with 4 mM l-glutamine, sodium bicarbonate (1.5 g liter⁻¹), glucose (4.5 g liter⁻¹), and 10% fetal bovine serum. Cells were seeded at 4 × 10⁶ in 3 ml medium per well in 24-well tissue culture plates and incubated at 37^oC with 5% CO₂ for 2 days (∼70% confluence) prior to infection. Each well was infected with a bacterium/DF-1 cell ratio of 10:1. For type 1 pilus induction, bacterial cultures were grown with two passages of 48 h each time in static LB with the appropriate antibiotics prior to infection. Growing bacteria on tryptic soy agar plates with the appropriate antibiotics induced P-type pilus expression. The experiment was performed with bacteria grown under both conditions with essentially similar results.

^cAdherence assays were performed essentially by the method of Elsinghorst (13). Just before infection, the medium in each well was replaced with 3 ml of fresh prewarmed cell culture medium. Twenty microliters of bacterial suspension was added to each well to attain a multiplicity of infection of 10. The plates were centrifuged at 600 × g for 5 min and incubated at 37^oC for 2 h. After 2 h, one set of triplicate wells was lysed by the addition of 20 μl of 5% Triton X-100. Bacteria present in the lysates, representing the total number of bacteria (intra- and extracellular), were enumerated by plating.

^dTo determine invasion frequencies, after the initial 2-h incubation, an additional set of wells was washed with PBS (Mg²⁺/Ca²⁺) and incubated for another 4 h in medium containing 100 μg ml⁻¹ of the membrane-impermeable bactericidal antibiotic gentamicin to kill adhered extracellular bacteria. Wells were washed three times with PBS, lysed with 1 ml 0.1% Triton X-100, and plated in various dilutions.

^eTo measure adherence, a second set of wells was washed five times with PBS supplemented with 2 mM MgCl₂ and CaCl₂, lysed with 1 ml of 0.1% Triton X-100, and plated in various dilutions. "Only adherence" was calculated as the numbers of bacteria recovered after PBS wash subtracted from the total number of bacteria present in each well.

^fAttachment index is calculated as follows: (CFU/ml of only adherent bacteria)/(total bacterial inoculum [CFU/ml]).

^gInvasion index was determined as the number of bacteria surviving incubation with gentamicin divided by the total number of bacteria present just before the addition of gentamicin, i.e., the only adherent population.