TABLE 3.
Attributes of APEC strain χ7122 and various isogenic mutants to colonize 12-day-old chicken embryos, invade internal organs, and disseminate in allantoic and amniotic fluids after 24 h and 48 h of infection
| Strain | Genotype | Aerobactin productiona | Hemolysin productionb | Serum resistancec [log10 (CFU/ml)] at the following time after infection:
|
Tissued | Tissue infectivitye [log10 (CFU/ml or mg of tissue) at the following time after infection:
|
||
|---|---|---|---|---|---|---|---|---|
| 0 h | 4 h | 24 h | 48 h | |||||
| χ7122 | Wild type | + | + | 8.6 ± 0.9 | 8.9 ± 0.9 | CAM | 2.2 ± 1.2 | 3.1 ± 1.5 |
| ALF | 2.0 ± 1.2 | 3.4 ± 1.0 | ||||||
| AMF | 2.2 ± 1.2 | 3.1 ± 1.6 | ||||||
| Liver | 3.8 ± 1.9 | 3.9 ± 1.2 | ||||||
| Spleen | 2.0 ± 1.9 | 3.2 ± 1.8 | ||||||
| Lungs | 3.3 ± 1.9 | 3.8 ± 1.8 | ||||||
| SM3000 | barA | −/+ | −/+ | 8.5 ± 0.9 | 5.0 ± 0.4 | CAM | 2.1 ± 1.9 | 3.2 ± 1.5 |
| ALF | 2.0 ± 0.7 | 2.0 ± 1.3 | ||||||
| AMF | 2.0 ± 1.0 | 2.1 ± 1.5f,g | ||||||
| Liver | 3.1 ± 1.4f,g | 2.1 ± 1.0 | ||||||
| Spleen | <1 ± 0.1f,g | <1 ± 0.1f,g | ||||||
| Lungs | 1.8 ± 0.8f,g | <1 ± 0.1f,g | ||||||
| M3001 | uvrY | −/+ | −/+ | 8.1 ± 0.8 | 4.1 ± 0.9 | CAM | 2.2 ± 1.2 | <1 ± 0.1f,g |
| ALF | 1.8 ± 1.0f,g | <1 ± 0.1f,g | ||||||
| AMF | <1 ± 0.1f,g | <1 ± 0.1f,g | ||||||
| Liver | 3.3 ± 1.4f,g | 1.5 ± 1.2f,g | ||||||
| Spleen | <1 ± 0.1f,g | <1 ± 0.1f,g | ||||||
| Lungs | 2.1 ± 1.3f,g | <1 ± 0.1f,g | ||||||
| SM3004 | uvrY/p-uvrY | + | + | 8.4 ± 0.8 | 9.1 ± 0.9 | CAM | 2.7 ± 0.9 | 3.2 ± 1.3 |
| ALF | 2.1 ± 1.3 | 2.0 ± 0.9 | ||||||
| AMF | 3.6 ± 1.4 | 3.5 ± 1.0 | ||||||
| Liver | 3.5 ± 1.4 | 3.6 ± 1.5 | ||||||
| Spleen | 3.2 ± 1.4 | 3.5 ± 1.5 | ||||||
| Lungs | 3.2 ± 1.3 | 3.5 ± 1.5 | ||||||
Aerobactin production was determined by cross-feeding E. coli K-12 LG1522 (63). Lawns of LG1522 were plated onto M-9 minimal agar medium containing 40 μg/ml of tryptophan and 200μg/ml of 2,2′-dipyridyl. Fresh bacterial cultures in LB of 107 cells in 20 μl PBS were spotted onto the lawn of the aerobactin indicator strain LG1522 and incubated for 18 h at 37oC. Growth of the indicator organism around the spots in a halo indicated aerobactin production denoted by a + sign. E. coli LG1315 was used as a positive control (L. K. Nolan), and E. coli HB101 and DH5α were used as negative controls. A + sign indicates growth of reporter strain LG1522 comparable to that of LG1315; a −/+ sign indicates growth of reporter strain is less than that seen around the positive control, while a − sign indicates no growth of the reporter strain.
Hemolysin activity was measured by spotting 107 bacterial cells in 20 μl of PBS (and also by streaking) onto sheep blood agar plates and incubating the cells for 18 h at 37oC. The appearance of the zone of erythrocyte lysis around or under the bacterial colonies indicated hemolysis. E. coli K-12 DH5α was used as a negative control, and DH5α carrying plasmid pSF4000 (67) was used as a positive control. Two natural field isolates, VMRCVM-APEC isolate 10 (hly+) and isolate 11 (hly mutant), were used as APEC-specific controls. Marginal differences between the wild type and various mutants are denoted (−/+), but spectrophotometric measurement of heme release from sheep erythrocytes did not exhibit a statistically significant difference between the wild-type strain and various mutants.
Serum bactericidal assay was measured by the ability of 107 CFU/ml of freshly grown bacteria in 100-μl volume to survive in 300 μl of 10% fresh normal chicken serum (obtained from blood collected by wing vein puncture of 4- to 5-week-old chickens). Viable cell counts were done just before addition of bacteria to serum (time zero) and at 1 h and 3 h after incubation. Bacterial serum sensitivity was defined as a 2-log-unit decrease in the number of viable bacteria. Serum-sensitive (DH5α) and serum-resistant (VMRCVM-APEC isolate 20, a natural field isolate) E. coli strains were used as negative and positive controls, respectively. Results are presented as mean values of two independent experiments ± standard deviations of the means.
CAM, chorioallantoic membrane; ALF, allantoic fluid; AMF, amniotic fluid.
The persistence of wild-type APEC and various mutants was determined by the chicken embryo lethality assay followed by enumerating bacterial counts in various organs of 12-day-old embryos (42). The allantoic cavities of eight embryonated eggs were inoculated with 100 μl of 5 × 103 CFU of each bacterial culture in PBS. At 24 h and 48 h, the eggs were candled for viability. Half of the embryos inoculated with E. coli χ7122 (wild type) and SM3004 (uvrY/p-uvrY) had died by 24 h, and the other half showed morbidity. At 48 h, most of the morbid embryos died. At a given time, the dead embryos were chosen for dissection. Tissues or fluids were aseptically removed from each embryo, weighed, homogenized in 2-ml microcentrifuge tubes, and centrifuged at 1,000 rpm for 5 min at room temperature. Three serial dilutions of the supernatant were plated in triplicate onto LB plates with appropriate antibiotics and incubated at 37oC for 18 h, and the bacterial load was determined in log10 CFU per mg (tissue or organ) or ml (allantoic and amniotic fluids) ± standard deviation for four embryos from each group.
This value is significantly different (P < 0.05) from the value for the wild-type strain.
This value is significantly different (P < 0.05) from the value for the complemented strain.