RESEARCH Antimicrobial Resistance Markers of Class 1 and Class 2 Integron- bearing Escherichia coli from Ir rigation Water and Sediments Matthew T. Roe,* Everardo Vega,* and Suresh D. Pillai* Municipal and agricultural pollution affects the Rio Grande, a river that separates the United States from Mexico. Three hundred and twenty-two Escherichia coli isolates were examined for multiple antibiotic resistance phenotypes and the prevalence of class 1 and class 2 inte- gron sequences. Thirty-two (10%) of the isolates were resistant to multiple antibiotics. Four (13%) of these iso- lates contained class 1–specific integron sequences; one isolate contained class 2 integron–specific sequences. Sequencing showed that the class 1 integron–bearing strain contained two distinct gene cassettes, sat-1 and aadA. Although three of the four class 1 integron–bearing strains harbored the aadA sequence, none of the strains was phenotypically resistant to streptomycin. These results suggest that integron-bearing E. coli strains can be present in contaminated irrigation canals and that these isolates may not express these resistance markers. Iantimicrobial resistance alleles by lateral gene transfer ntegron gene sequences contribute to the spread of of gene cassettes in a variety of enteric bacteria, including Campylobacter spp., Escherichia coli, and Salmonella enterica serotype Typhimurium (1–4). The gastrointestinal environment is suspected of serving as a reservoir for inte- gron-bearing strains; when antimicrobial exposure occurs, gene transfer events—which spread cassettes between commensal organisms that are expelled into the environ- ment (2)—would also occur. The Rio Grande, the river separating the United States from Mexico along the Texas-Mexico region, serves as a source for irrigation water in Texas and Mexico. Previous studies in our laboratory and others have shown that the transboundary region is subject to extensive microbial and chemical contamination. This contamination has been associated with agricultural, municipal, and industrial wastes originating from both sides of the border (5,6). Leaking septic tanks and wastewater effluent discharges result in fecal contamination levels as high as 2,000 CFU/mL of fecal coliforms (7,8). *Texas A&M University, College Station, Texas, USA Because of the strategic importance of the Rio Grande for U.S. agriculture and the potential transmission of antimicrobial resistance determinants by means of food crops, we investigated the prevalence and characteristics of class 1 and class 2 integron–bearing E. coli strains. These strains were previously isolated from a study inves- tigating fecal contaminants in irrigation water and associ- ated sediments at specific locations along the river (9). Methods Three hundred and twenty-two E. coli isolates were previously isolated from irrigation water and associated sediments at the El Paso, Presidio, and Weslaco regions of the river (9). After being confirmed as E. coli by MUG (4- methyl umbelliferyl-β-D-glucoronide)–based fluores- cence, these isolates were screened for antimicrobial sus- ceptibility by using the agar dilution method (10,11). The isolates were tested against ampicillin, tetracycline, ceftri- axone, cephalothin, gentamicin, kanamycin, streptomycin, chloramphenicol, ciprofloxacin, and trimethoprim/sul- famethoxazole. The antibiotics were tested at concentra- tions established by the National Antimicrobial Resistance System (12). Isolates that were multidrug resistant (resistant to two or more antimicrobial agents) were grown overnight in 5 mL of Mueller-Hinton broth (Accumedia, Baltimore, MD) with the appropriate concentration of antimicrobial com- pound. A 1-mL aliquot of the culture was centrifuged at 10,000 rpm for 2 min. The cell pellet was resuspended in 500 µ L of sterile water and boiled for 10 min. The result- ing DNA suspension was used as template DNA in poly- merase chain reaction (PCR) amplification for the class 1 and class 2 integrase gene and variable regions using the primer sequences shown in the Table (13–15). The PCR reactions used 10 µL of template DNA, 5 µM of primers, 25 mM MgCl, 10 mM deoxynucleotide triphosphate, and 23 ng bovine serum albumin. Nuclease- free water (Ambion, Austin, TX) was added to achieve a volume of 50 µ L. A “hot start” method was used, and 1.25 U of Taq DNA polymerase (Sigma, St. Louis, MO) was 822 Emerging Infectious Diseases • Vol. 9, No. 7, July 2003