These images show two laboratory tools for diagnosis of enteroaggregative Escherichia coli (EAEC). The adherence assay, which assesses the ability of EAEC cells to adhere in a honeycomb pattern to both the glass coverslip and to eukaryotic cultured cells, and the polymerase chain reaction technique in which hallmark genes of EAEC are amplified.
Enteroaggregative Escherichia coli has been recognized as an etiologic agent of acute and persistent diarrhea among children, travelers, and in human immunodeficiency virus-infected persons (7). This enteric bacterium can be diagnosed by means of the adhesion assay, considered as the “gold standard” (3, 6), but the test is expensive and is a time and labor intensive method. A specific, sensitive, and less time-consuming technique is the multiplex polymerase chain reaction (mPCR), in which two or more sets of primer pairs specific for different targets are introduced in the same tube (11, 12). Several authors have developed mPCR techniques in order to amplify different loci of EAEC, contributing to a more rapid diagnosis of this pathogenic variant of diarrheagenic E. coli (1, 4).
Figure 1A shows the typical aggregative adherence pattern of the EAEC prototype strain E. coli 042 on SW-480 cells (human colon adenocarcinoma cell line) as well as on the glass coverslip. In this adhesion pattern, aggregates of bacteria assumed a hallmark “stacked-brick” or “honeycomb” pattern on the surface of the cells, as well as on the glass coverslip. E. coli ATCC 25922, which is unable to adhere to both the glass slide and the SW-480 cells, was used as a negative control (Fig. 1B). The adhesion assay was performed with monolayers of SW-480 cells grown on circular coverslips in wells of 24-well tissue culture plates. Thereafter, the carcinogenic cells were infected for 3 hours at 37°C with 107 to 108 bacteria/ml, previously plated overnight at 37°C in 1% tryptone broth with 1% D-mannose. After the infection period, the coverslips were washed 3 times with phosphate-buffered saline, fixed with 70% methanol, and stained with 10% Giemsa (3). The stained coverslips were examined with a light microscope under oil immersion microscopy (total magnification 1,000X).
Figure 2 depicts an electrophoretic profile of three plasmid-borne genes of the same prototype EAEC strain, amplified by an mPCR reaction using three primer sets (aap, aggR, and AA probe genes). Lanes: 1, 100-bp DNA ladder (molecular weight marker); 2, no PCR products obtained from DNA of E. coli ATCC 25922 (negative control); 3, PCR products obtained from the E. coli 042 strain; 4 to 6, PCR products obtained with 042 strain and primers for the AA probe, aggR, and aap genes, respectively. All PCR reactions were prepared according to Cerna, et al (1). The amplicons were separated by electrophoresis on 2.5% agarose gel in Tris-acetate-EDTA buffer and visualized under a UV-transilluminator after staining with 0.5 mg/ml of ethidium bromide solution. The picture was obtained using a Canon-EOS Rebel T2i camera.
EAEC is considered as an emerging pathogen and reports about the diarrhea associated with this bacterium have been growing (7). The adherence test remains the gold standard technique, but it is only performed in reference laboratories around the world; thus, several authors have developed molecular methods to detect specific genes of EAEC (1, 5, 8, 13), such as those shown here. This mPCR detects the typical loci found among the most virulent EAEC strains (10) and has been used as a laboratory tool in epidemiologic studies for a rapid diagnosis of EAEC (1, 2, 9).
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