Abstract
Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [14C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.
One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).
Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.
This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.
MATERIALS AND METHODS
Bacterial strains.
For the experiments on lactate production, three E. coli isolates from children with acute diarrhea were chosen as index strains after their in vitro adherence to HeLa cells had been tested by the method of Nataro et al. (20). One isolate displayed diffuse adherence, and one was nonadherent. The third strain adhered in a localized manner and on serotyping was found to belong to O127K63 (Wellcome Diagnostics). None of the three strains hybridized with enzyme-labelled oligonucleotide probes (Du Pont) that detect heat-labile and heat-stable enterotoxins, and none demonstrated verocytotoxicity in cell culture assays with Vero cells. Three well-characterized enteropathogenic E. coli strains were used as controls: E2348/69, CVD206, and JPN15. E2348/69 and CVD206 are an isogenic pair consisting of a wild-type, adherent (localized) strain and its attaching and effacing lesion-deficient (eaeA) mutant, respectively. JPN15, a derivative of E2348/69, has a depletion of the E. coli adherence factor plasmid usually associated with initial adherence. To assess the consistency of d-lactate production by adherent strains, 18 additional isolates from children with acute diarrhea were also studied. Five E. coli isolates displayed diffuse adherence, eight showed localized adherence, and five showed aggregative adherence.
Removal of succinic acid from the medium was tested in the three index strains.
Measurement of pH, lactate, and bacterial growth.
The E. coli strains were cultured overnight at 37°C in tryptone water and diluted with phosphate-buffered saline (PBS) to yield a concentration of 1 × 108 to 2 × 108 bacteria/ml. Eagle’s minimum essential medium (MEM) containing 1% d-mannose was purged with the appropriate gas mixture and dispensed into 48-well Costar tissue culture clusters. A 510-μl volume of MEM-mannose was inoculated with 20 μl of dilute E. coli suspension. Bacteria were cultured for 0, 1, 2, 3, 4, or 5 h at 37°C in 5% CO2–95% air, 5% CO2–95% O2, or 5% CO2–95% N2. pH was measured at the end of each incubation period with a pH meter (Radiometer) using a pH microelectrode with a sensitivity range of pH 0 to 14 and 0 to 80°C. Following incubation, the culture medium was collected and centrifuged twice in a Beckman Microfuge. The E. coli-free supernatant fluid (SNF) was used for enzymatic analysis of lactic acid. Each individual assay was carried out three times.
Assays for d- and l-lactic acid were performed with a commercial kit (catalog no. 149993; Boehringer Mannheim) with the following modifications to the manufacturer’s recommended method. For the determination of d-lactate levels, d-lactate dehydrogenase was added (catalog no. 10694; Boehringer Mannheim) at a concentration of 75 U/ml. The sample volume was increased fourfold. Final absorbances were read at 340 nm after 25 min to ensure that the reaction had run to completion. For the measurement of l-lactate, the sample volume was increased 40-fold. The sensitivity and linearity of the assay were established with a commercial l-lactate standard (catalog no. 125440; Boehringer Mannheim) over a concentration range of 5 to 100 μmol/liter. Bacterial concentrations in cultures before and after incubation were estimated by measuring the optical density (OD) at 600 nm, where 1 OD unit is 8 × 108 cells/ml. The viability of bacterial suspensions was measured by surface colony counts on agar plates.
To check the effect of depletion of the medium, E2348/69 was incubated in MEM-mannose in 5% CO2–95% air as described above for 2 h. The bacteria and medium were separated by centrifugation. The pellet was resuspended in either fresh medium or the medium in which it had been cultured for the previous 2 h. After a further 2 h of incubation the d-lactate concentration was measured. The experiment was done in triplicate.
Removal of succinic acid from the medium.
The HeLa cell adherence assay was performed with monolayers of HeLa cells as described by Nataro et al. (20). Briefly, 48-well tissue culture clusters (Costar) were seeded with HeLa cells at 105/ml. The cells were cultured overnight at 37°C in 5% CO2 in MEM supplemented with 2.2 g of NaHCO3/liter, 10% fetal calf serum, 30 μg of penicillin/ml, and 50 μg of streptomycin/ml. E. coli test strains were grown overnight in tryptone water and subsequently diluted with PBS (pH 7.4) to a concentration of 1 × 108 to 2 × 108 bacteria/ml. Semiconfluent monolayers of HeLa cells were washed, and 500 μl of MEM containing 1% d-mannose was added to each well. Twenty microliters of the diluted E. coli suspension and 10 μl of [14C]succinic acid (approximately 5,000 cpm) were added to HeLa cells. The succinic acid tracer was either [2,3-14C]succinic acid (code CFA 142) or [1,4-14C]succinic acid (code CFA 66; Radiochemical Centre, Amersham, United Kingdom).
The [14C]succinic acid tracer studies were carried out in the absence and presence of HeLa cells and in the absence and presence of adherent or nonadherent E. coli. All samples were analyzed in duplicate. Culture medium, E. coli, and HeLa cells were harvested at the beginning of the incubation period (0 h) and after 3 h of incubation. Culture medium and E. coli were aspirated off and centrifuged in a Beckman Microfuge for 5 min. [14C]succinic acid was extracted from the SNF. The E. coli pellet was washed once and resuspended in PBS. Wells containing HeLa cells were also washed once with PBS. The cells were dissociated with trypsin-EDTA and resuspended in PBS.
From all SNFs, methyl derivatives of [14C]succinic acid were prepared and extracted into chloroform (10). Aliquots of the chloroform extracts as well as samples of the water phase after extraction were assessed for 14C counts. The amount recovered was the sum of the two counts. Direct 14C counting was performed on the HeLa cells and E. coli suspensions.
In the calculations of recovery of 14C, counts in the SNFs, HeLa cells, and E. coli were expressed as percentage of the total counts added to each well, i.e., disintegrations per minute from 10 μl of [14C]succinic acid in 520 μl of culture medium. The mean percent recoveries in SNF and cells were obtained from three separate experiments using [2,3-14C]succinic acid and from five using [1,4-14C]succinic acid.
The difference in the percent recovery at the beginning and after 3 h of incubation was calculated for each E. coli SNF and its matching cellular component. The amount lost, presumably as CO2, was calculated from the difference between the percentage of label removed from the SNF and that recovered in the cellular compartment.
Statistical analysis.
Analysis of variance was used to test for differences in lactate production, pH, and bacterial growth. The succinic acid data was analyzed by analysis of variance in a 23 factorial design (25) to determine the significance of each factor and interactions between them. The three factors were time, HeLa cells, and E. coli, and the two levels were the presence and absence of each factor.
RESULTS
Lactate production and pH measurements.
No detectable l-lactate was produced by any of our index isolates, but all produced d-lactate.
Tables 1 and 2 summarize the mean d-lactate levels and pH values for our localized adherent, diffuse adherent, and nonadherent E. coli strains and for E2348/69, CVD206, and JPN15 in 5% CO2 and 95% air.
TABLE 1.
d-Lactate production by adherent and nonadherent E. coli strains in 5% CO2–95% air
| Time (h) | Concn of d-lactate (μmol/liter) produced bya:
|
|||||
|---|---|---|---|---|---|---|
| Index strains
|
Control strains
|
|||||
| Diffuse adherent | Nonadherent | Localized adherent | E2348/69 | JPN15 | CVD206 | |
| 0 | 42.10 (10.61) | 29.88 (9.80) | 44.82 (8.49) | 10.87 (5.93) | 16.29 (5.88) | 0.67 (0.67) |
| 1 | 51.61 (8.91) | 48.89 (6.23) | 51.61 (7.57) | 11.54 (9.58) | 12.22 (5.39) | 2.71 (1.79) |
| 2 | 62.48 (12.08) | 57.04 (6.22) | 53.69 (5.34) | 9.50 (7.57) | 11.54 (4.45) | 0.00 (0.00) |
| 3 | 78.78 (9.80) | 63.84 (17.35) | 52.30 (2.44) | 26.48 (6.55) | 9.50 (4.90) | 2.03 (2.03) |
| 4 | 207.82 (14.70) | 122.25 (18.84) | 76.06 (7.19) | 97.11 (7.84) | 9.50 (4.45) | 4.75 (2.44) |
| 5 | 926.11 (68.02) | 433.91 (114.23) | 114.10 (9.42) | 472.69 (78.64) | 17.65 (7.83) | 0.69 (0.68) |
Values are means; values in parentheses are SEs.
TABLE 2.
pH of medium in 5% CO2–95% air
| Time (h) | Mean pH of medium (SE) of:
|
|||||
|---|---|---|---|---|---|---|
| Index strains
|
Control strains
|
|||||
| Diffuse adherent | Nonadherent | Localized adherent | E2348/69 | JPN15 | CVD206 | |
| 0 | 7.50 (0.04) | 7.50 (0.04) | 7.52 (0.07) | 7.66 (0.04) | 7.70 (0.05) | 7.70 (0.04) |
| 1 | 7.50 (0.04) | 7.50 (0.04) | 7.50 (0.04) | 7.37 (0.03) | 7.43 (0.03) | 7.49 (0.05) |
| 2 | 7.50 (0.04) | 7.50 (0.04) | 7.50 (0.04) | 7.30 (0.04) | 7.38 (0.03) | 7.41 (0.04) |
| 3 | 7.17 (0.07) | 7.30 (0.14) | 7.42 (0.12) | 6.87 (1.71) | 7.24 (0.14) | 7.23 (0.16) |
| 4 | 6.57 (0.07) | 6.90 (0.04) | 7.10 (0.04) | 6.41 (0.09) | 7.14 (0.07) | 7.22 (0.10) |
| 5 | 5.60 (0.29) | 6.45 (0.04) | 6.62 (0.02) | 5.84 (0.12) | 7.01 (0.07) | 7.17 (0.09) |
The trends in pH decreases and d-lactate increases in our localized adherent strain and in E2348/69 were similar. Very little d-lactate could be detected up to 2 h of incubation. By 3 h d-lactate levels had started to rise and pH had started to fall. A further progressive increase in d-lactate levels and drop in pH occurred between 3 and 5 h (Tables 1 and 2). At 4 and 5 h d-lactate levels were higher in E2348/69 than in our localized adherent strain. Unlike the parent strain, E2348/69, JPN15, which lacks the E. coli adherence factor plasmid, produced very small amounts of d-lactate, and CVD206, the eae mutant, produced only trace amounts during the entire 5-h incubation period. The pH drop was small in CVD206 and JPN15, and pHs remained above 7.0 at the end of 5 h of incubation.
Our diffuse and nonadherent isolates produced similar concentrations of d-lactate up to 3 h of incubation, but at 4 and 5 h, the diffuse adherent isolate showed approximately twice as much d-lactate as the nonadherent strain. With both strains, concentrations of d-lactate were higher than those found with the localized adherent isolate.
The amount of d-lactate produced by our three index isolates was not influenced by the introduction of different gases during culture periods of up to 3 h. In 5 h, the diffuse adherent isolate produced the same amount of d-lactate under anaerobic and aerobic conditions (1,145 ± 108 and 1,140 ± 81 μmol/liter, respectively) and a smaller amount in air (Table 1). With the nonadherent strain, the pattern was similar, with 918 ± 81 μmol/liter being produced in N2, 992 ± 49 μmol/liter being produced in O2, and less being produced in air (Table 1). In N2 and O2, the localized adherent isolate produced amounts of d-lactate similar to that produced in 5% CO2–95% air (Table 1). The magnitudes of the increase in d-lactate levels and the drop in pH were consistent for each isolate regardless of culture conditions.
The increase in lactate concentration was not due to depletion of the medium. The mean lactate level of 93.7 μmol/liter (standard error [SE], 6.1) for E2348/69 resuspended in the medium in which it had been cultured for the first 2 h was similar to the 97.1 μmol/liter (SE, 7.8) found after 4 h of standard incubation (Table 1). The concentration was higher when the strain was resuspended in fresh medium, 158.9 μmol/liter (SE, 6.2).
Table 3 shows d-lactate concentrations obtained from the 18 additional isolates. At the end of the 5-h incubation period, the highest levels were obtained in the diffuse adherent group, whereas the lowest concentrations were detected in the localized adherent category. This is consistent with d-lactate values shown by the two index strains with these adherence patterns. Isolates in the aggregative adherent group showed lower levels than the diffuse adherent strains but higher levels than the localized adherent strains.
TABLE 3.
d-Lactate production by 18 additional E. coli isolates in 5% CO2–95% air
| Time (h) | Concn of d-lactate (μmol/liter) produced by strain typea
|
||
|---|---|---|---|
| Diffuse adherent (n = 5) | Localized adherent (n = 8) | Aggregative adherent (n = 5) | |
| 0 | 7.33 (2.62) | 6.11 (2.30) | 4.48 (2.68) |
| 1 | 12.22 (2.80) | 6.87 (2.17) | 5.70 (3.37) |
| 2 | 12.22 (3.91) | 9.41 (2.07) | 11.81 (1.49) |
| 3 | 110.02 (28.86) | 87.60 (9.81) | 151.10 (20.45) |
| 4 | 328.43 (70.45) | 124.02 (10.90) | 207.00 (27.92) |
| 5 | 958.43 (103.78) | 375.40 (44.81) | 703.74 (110.28) |
Values are means; values in parentheses are SEs.
Bacterial growth.
Bacterial concentrations measured by OD correlated well with viability counts. All isolates displayed active growth within 0.5 h at 37°C. Active multiplication continued throughout the 5 h of incubation (Table 4), whereas pH changes occurred only after 2 h (Table 2).
TABLE 4.
Growth of control strains in MEM-mannose
| Time (h) | Mean count (108 cells/ml) ofa:
|
||
|---|---|---|---|
| E2348/69 | JPN15 | CVD206 | |
| 0 | 0.031 (0.001) | 0.138 (0.007) | 0.019 (0.001) |
| 1 | 0.267 (0.005) | 0.162 (0.004) | 0.122 (0.002) |
| 2 | 0.886 (0.017) | 0.439 (0.017) | 0.366 (0.038) |
| 3 | 2.087 (0.013) | 0.767 (0.019) | 0.529 (0.002) |
| 4 | 4.818 (0.036) | 2.034 (0.072) | 0.865 (0.039) |
| 5 | 5.714 (0.015) | 3.086 (0.057) | 1.199 (0.026) |
Values in parentheses are SEs.
Removal of succinic acid.
Table 5 summarizes the mean changes in recovery and SEs when [2,3-14C]succinic acid was used as the tracer. The decrease in recovery from the SNF was smallest with the localized adherent strain of E. coli (3.33%) and greatest with the nonadherent strain (12.44%). The diffuse adherent E. coli strain had decreased recovery (8.99%). There was no interaction between the HeLa cells and either the diffuse adherent or the nonadherent strains, but there was a significant interaction between the localized adherent strain and the HeLa cells. Incorporation into E. coli varied between 2.81% for the nonadherent strain and 3.68% for the diffuse adherent strain. With the localized adherent strain most of the tracer removed from the SNF (3.33%) was recovered from the E. coli cells (3.05%). There were much larger differences in tracer recovery (i.e., tracer lost, presumably as CO2) with the diffuse adherent strain (SNF, 8.99%, and E. coli, 3.05%) and nonadherent strain (SNF, 12.44%, and E. coli, 2.81%).
TABLE 5.
Change in percent recovery after 3 h of incubation of E. coli with [2,3-14C]succinic acid
| Sample type and cells incubated | Change in % recovery of strain typea
|
||
|---|---|---|---|
| Localized adherent | Diffuse adherent | Nonadherent | |
| SNF | |||
| HeLa | +0.19 (0.96) | −1.31 (1.93) | −1.31 (1.93) |
| E. coli | −3.33 (1.11)b | −8.99 (0.78)b | −12.44 (4.54)b |
| HeLa + E. coli | −9.75 (0.17)c | −8.99 (0.21) | −11.84 (5.28) |
| Cells | |||
| HeLa | +0.01 (0.06) | −0.10 (0.13) | −0.10 (0.13) |
| E. coli | +3.05 (1.09)b | +3.68 (0.12)b | +2.81 (0.68)b |
| HeLa + E. coli | +3.02 (0.79) | +4.28 (0.20)c | +3.08 (0.98) |
Values are means; values in parentheses are SEs.
Significant difference between values in a row (P < 0.05).
Significant interaction between the E. coli strain and HeLa cells (P < 0.05).
The mean recoveries with [1,4-14C]succinic acid was used as the tracer are shown in Table 6. The pattern was the same as that seen with [2,3-14C]succinic acid. Recovery from SNF was greatest with the localized adherent strain and lowest with the nonadherent strain, with no interaction between any of the E. coli strains and the HeLa cells. Removal of [1,4-14C]succinic acid from the SNF was much greater than removal of [2,3-14C]succinic acid, but the amounts of 14C incorporated into the E. coli and HeLa cells were slightly lower.
TABLE 6.
Change in percent recovery after 3 h of incubation of E. coli with [1,4-14C]succinic acid
| Sample type and cells incubated | Change in % recovery of strain typea
|
||
|---|---|---|---|
| Localized adherent | Diffuse adherent | Nonadherent | |
| SNF | |||
| HeLa | −3.03 (1.41) | −3.90 (3.31) | −5.12 (3.17) |
| E. coli | −9.35 (1.27)b | −17.40 (2.51)b | −23.39 (2.03)b |
| HeLa + E. coli | −8.31 (1.39) | −18.09 (3.73) | −23.34 (3.01) |
| Cells | |||
| HeLa | +0.05 (0.03) | −0.06 (0.04) | −0.01 (0.05) |
| E. coli | +0.86 (0.08)b | +2.52 (0.31)b | +1.20 (0.30)b |
| HeLa + E. coli | +1.08 (0.13) | +2.63 (0.58) | +1.09 (0.33) |
Values are means; values in parentheses are SEs.
Significant difference between values in a row (P < 0.05).
The difference between the amount of label removed from the SNF and the amount recovered from the cells was greatest with nonadherent E. coli, less with the diffuse adherent strain, and least with the localized adherent isolate. The pattern was the same for both tracers, although all losses were greater with [1,4-14C]succinic acid than [2,3-14C]succinic acid (Table 7).
TABLE 7.
Differences in removal of label from the SNF and recovery in the cellular compartment
| Strain type | Difference in % removed from SNF and % recovered in cellsa
|
|
|---|---|---|
| [2,3-14C]succinic acid (n = 3) | [1,4-14C]succinic acid (n = 5) | |
| Localized adherent | −6.38 (1.74) | −10.22 (1.34) |
| Diffuse adherent | −12.67 (0.83) | −19.91 (2.65) |
| Nonadherent | −15.25 (5.15) | −24.59 (2.09) |
i.e., the amount presumably lost as CO2. Values are means; values in parentheses are SEs. n, number of experiments.
DISCUSSION
It is widely accepted that the adherence of enteropathogenic E. coli to intestinal epithelial cells in infants is associated with the production of diarrhea. However, information which elucidates actual metabolic events during bacterial adhesion remains incomplete. Our previous investigation into E. coli-induced pH changes pointed towards a difference in metabolic behavior between adherent and nonadherent E. coli strains during their adherence to HeLa cells. This study confirmed that the amounts of d-lactate produced by our localized adherent, diffuse adherent, and nonadherent E. coli index strains after 3 h of incubation were not the same. The fact that d-lactate concentrations differ in isolates displaying different adherence patterns was also shown with 18 additional, freshly collected isolates. Mean d-lactate concentrations in the three groups were not the same. Further support for the postulate that the E. coli strains are inherently different comes from the very low d-lactate levels detected in CVD206 and JPN15, two genetically engineered strains, whose adherence patterns no longer resemble that of the parental, localized adherent strain, E2348/69. This finding suggests a difference in metabolism between localized adherent, diffuse adherent, aggregative adherent, and nonadherent isolates.
d-Lactate production by the E. coli strains coincided with pH decreases in tissue culture medium. For example, with E2348/69, the increase in d-lactate concentration coincided with a decrease in pH between 2 and 5 h of incubation. In contrast, the low d-lactate levels found with CVD206 and JPN15 corresponded to minimal changes in pH over the same period.
The pattern of lactate production and the pH changes cannot be explained purely by bacterial growth. Lactate levels and pH showed little change in the first 2 h, while bacterial counts increased by a factor of about 30. Between 3 and 5 h, lactate production of some strains led to a 10-fold or greater increase in lactate concentration and the colony counts increased two to four times. Further differences in growth rates of isolates with different adherence phenotypes do not account for the differences in lactate production. For example, when the lactate concentration is expressed in terms of colony count instead of per liter, E2348/69 produced about 82.7 μmol, JPN15 produced 5.7 μmol, and CVD206 produced 0.6 μmol per 108 colonies at 5 h.
HeLa cells did not remove [14C]succinic acid from the tissue culture medium irrespective of whether E. coli adhered, but the E. coli removed significant amounts of [14C]succinic acid from the SNF; the amount varied from strain to strain. The nonadherent strain removed the largest amount of [2,3-14C]succinic acid, and the localized adherent isolate removed the smallest amount. Uptake of the tracer by all three E. coli isolates was low but significant, the greatest percentage being recovered from diffuse adherent E. coli. The amount of tracer presumed lost as CO2 differed among the isolates used. When [1,4-14C]succinic acid was used as the tracer there was the same order in the amount of label removed from the medium by the three E. coli isolates. However, larger quantities of [1,4-14C]succinic acid than [2,3-14C]succinic acid were removed by all isolates. This indicates that the metabolism of [14C]succinic acid by E. coli occurs first at the terminal carbon positions.
The pH changes previously observed when adherent and nonadherent E. coli strains were incubated in the presence of HeLa cells cannot be attributed to the removal of organic acids by HeLa cells or to interactions between the E. coli and HeLa cells. Rather, the E. coli cells removed [14C]succinic acid from the culture medium, and the amounts removed by nonadherent E. coli differed from those removed by adherent bacteria. This is another metabolic difference between attaching and nonattaching isolates. The previous studies into E. coli-induced pH changes showed a greater drop in pH in tissue culture medium derived from adherent E. coli than in that from nonadherent E. coli. The metabolic difference observed then, in the form of pH differences, is confirmed by the different metabolic activities in the production and removal of organic acids from the culture medium during incubation.
From our studies we conclude that inherent metabolic differences between adherent and nonadherent E. coli strains exist. These differences may play a role in the pathogenic mechanism.
ACKNOWLEDGMENTS
We express our sincere appreciation to J. P. Nataro of the Center for Vaccine Development, University of Maryland, Baltimore, for providing strains E2348/69, JPN15, and CVD206 and for helpful discussions in the course of this work.
REFERENCES
- 1.Baldwin T J, Brooks S F, Knutton S, Hernandez H A M, Aitken A, Williams P H. Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli. Infect Immun. 1990;58:761–765. doi: 10.1128/iai.58.3.761-765.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Canil M, Rosenshine I, Ruschkowoski S, Donnenberg M S, Kaper J B, Finlay B B. Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers. Infect Immun. 1993;61:2755–2762. doi: 10.1128/iai.61.7.2755-2762.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Clausen C R, Christie D L. Chronic diarrhoea in infants caused by adherent enteropathogenic Escherichia coli. J Pediatr. 1982;100:358–361. doi: 10.1016/s0022-3476(82)80429-0. [DOI] [PubMed] [Google Scholar]
- 4.Cravioto A, Gross R J, Scotland S M, Rowe B. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr Microbiol. 1979;3:95–99. [Google Scholar]
- 5.Cravioto A, Reyes R E, Ortega R, Fernández G, Hernández R, Lopéz D. Prospective study of diarrhoeal disease in a cohort of rural Mexican children: incidence and isolated pathogens during the first two years of life. Epidemiol Infect. 1988;101:123–134. doi: 10.1017/s0950268800029289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Cravioto A, Tello A, Navarro A, Ruiz J, Villafán H, Uribe F, Eslava C. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet. 1991;337:262–264. doi: 10.1016/0140-6736(91)90868-p. [DOI] [PubMed] [Google Scholar]
- 7.Embaye H, Hart C A, Getty B, Fletcher J N, Saunders J R, Batt R M. Effects of enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit intestinal mucosa. Gut. 1992;33:1184–1189. doi: 10.1136/gut.33.9.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Girón J A, Jones T, Millán-Velasco F, Castro-Muñoz E, Zárate L, Fry J, Frankel G, Moseley S L, Baudry B, Kaper J B, Schoolnik G K, Riley L W. Diffuse-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico. J Infect Dis. 1991;163:507–513. doi: 10.1093/infdis/163.3.507. [DOI] [PubMed] [Google Scholar]
- 9.Gomes T A T, Blake P A, Trabulsi L R. Prevalence of Escherichia coli strains with localized, diffuse, and aggregative adherence to HeLa cells in infants with diarrhea and matched controls. J Clin Microbiol. 1989;27:266–269. doi: 10.1128/jcm.27.2.266-269.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Holdeman L V, Cato E P, Moore W E C, editors. Anaerobe laboratory manual. 4th ed. Blacksburg: Virginia Polytechnic Institute and State University; 1977. p. 134. [Google Scholar]
- 11.Knutton S, Lloyd D R, McNeish A S. Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987;55:69–77. doi: 10.1128/iai.55.1.69-77.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Law D. Adhesion and its role in the virulence of enteropathogenic Escherichia coli. Clin Microbiol Rev. 1994;7:152–173. doi: 10.1128/cmr.7.2.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Levine M M. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic and enteroadherent. J Infect Dis. 1987;155:377–389. doi: 10.1093/infdis/155.3.377. [DOI] [PubMed] [Google Scholar]
- 14.Levine M M, Ferreccio C, Prado V, Cayazzo M, Abrego P, Martinez J, Maggi L, Baldini M M, Martin W, Maneval D, Kay B, Guers L, Lior H, Wasserman S S, Nataro J P. Epidemiologic studies of Escherichia coli diarrhoeal infections in a low socioeconomic level peri-urban community in Santiago, Chile. Am J Epidemiol. 1993;138:849–869. doi: 10.1093/oxfordjournals.aje.a116788. [DOI] [PubMed] [Google Scholar]
- 15.Levine M M, Prado V, Robins-Browne R, Lior H, Kaper J B, Moseley S L, Gicquelais K, Nataro J P, Vial P, Tall B. Use of DNA probes and HEp-2 cell adherence assay to detect diarrheagenic Escherichia coli. J Infect Dis. 1988;158:224–228. doi: 10.1093/infdis/158.1.224. [DOI] [PubMed] [Google Scholar]
- 16.Mathewson J J, Cravioto A. HEp-2 cell adherence as an assay for virulence among diarrheagenic Escherichia coli. J Infect Dis. 1989;159:1057–1060. doi: 10.1093/infdis/159.6.1057. [DOI] [PubMed] [Google Scholar]
- 17.McCabe K, Mann M D, Bowie M D. pH changes during in vitro adherence of Escherichia coli to HeLa cells. Infect Immun. 1994;62:5164–5167. doi: 10.1128/iai.62.11.5164-5167.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Moon H W, Whipp S C, Argenzio R A, Levine M M, Gianella R A. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983;41:1340–1351. doi: 10.1128/iai.41.3.1340-1351.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Nataro J P, Kaper J B, Robins-Browne R, Prado V, Vial P, Levine M M. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J. 1987;6:829–831. doi: 10.1097/00006454-198709000-00008. [DOI] [PubMed] [Google Scholar]
- 20.Nataro J P, Scaletsky I C A, Kaper J B, Levine M M, Trabulsi L R. Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli. Infect Immun. 1985;48:378–383. doi: 10.1128/iai.48.2.378-383.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rothbaum R, McAdams A J, Giannella R, Partin J C. A clinicopathologic study of enterocyte-adherent Escherichia coli: a cause of protracted diarrhea in infants. Gastroenterology. 1982;83:441–454. [PubMed] [Google Scholar]
- 22.Scaletsky I C A, Lourdes M, Silva M, Trabulsi L R. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun. 1984;45:534–536. doi: 10.1128/iai.45.2.534-536.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Scotland S M, Willshaw G A, Smith H R, Gross R J, Rowe B. Adhesion to cells in culture and plasmid profiles of enteropathogenic Escherichia coli isolated from outbreaks and sporadic cases of infant diarrhea. J Infect. 1989;19:237–249. doi: 10.1016/s0163-4453(89)90729-9. [DOI] [PubMed] [Google Scholar]
- 24.Sherman P, Drumm B, Karmali M, Cutz E. Adherence of bacteria to the intestine in sporadic cases of entero-pathogenic Escherichia coli-associated diarrhea in infants and young children: a prospective study. Gastroenterology. 1989;96:86–94. doi: 10.1016/0016-5085(89)90768-3. [DOI] [PubMed] [Google Scholar]
- 25.Snedecor G W, Cochran W G. Statistical methods. 6th ed. Ames: Iowa State University Press; 1967. pp. 359–361. [Google Scholar]
- 26.Taylor C J, Hart A, Batt R M, McDougall C, McLean L. Ultrastructural and biochemical changes in human jejunal mucosa associated with enteropathogenic Escherichia coli (O111) infection. J Pediatr Gastroenterol Nutr. 1986;5:70–73. doi: 10.1097/00005176-198601000-00013. [DOI] [PubMed] [Google Scholar]
- 27.Ulshen M H, Rollo J L. Pathogenesis of Escherichia coli gastroenteritis in man—another mechanism. N Engl J Med. 1980;302:99–102. doi: 10.1056/NEJM198001103020207. [DOI] [PubMed] [Google Scholar]
- 28.Vial P A, Mathewson J J, Du Pont H L, Guers L, Levine M M. Comparison of two assay methods of patterns of adherence to HEp-2 cells of Escherichia coli from patients with diarrhoea. J Clin Microbiol. 1990;28:882–885. doi: 10.1128/jcm.28.5.882-885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Vial P A, Robins-Browne R, Lior H, Prado V, Kaper J B, Nataro J P, Maneval D, Elsayed E, Levine M M. Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrhoeal disease. J Infect Dis. 1988;158:70–79. doi: 10.1093/infdis/158.1.70. [DOI] [PubMed] [Google Scholar]