Antimicrobial resistance markers of Class 1 and Class 2 integron-bearing Escherichia coli from irrigation water and sediments - Research

Emerging Infectious Diseases, July, 2003 by Matthew T. Roe, Everardo Vega, 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 coil isolates were examined for multiple antibiotic resistance phenotypes and the prevalence of class 1 and class 2 integron sequences. Thirty-two (10%) of the isolates were resistant to multiple antibiotics. Four (13%) of these isolates 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. coil strains can be present in contaminated irrigation canals and that these isolates may not express these resistance markers.

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Integron gene sequences contribute to the spread of antimicrobial resistance alleles by lateral gene transfer 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 integron-bearing strains; when antimicrobial exposure occurs, gene transfer events--which spread cassettes between commensal organisms that are expelled into the environment (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).

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 investigating fecal contaminants in irrigation water and associated 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-[beta]-D-glucoronide)-based fluorescence, these isolates were screened for antimicrobial susceptibility by using the agar dilution method (10,11). The isolates were tested against ampicillin, tetracycline, ceftriaxone, cephalothin, gentamicin, kanamycin, streptomycin, chloramphenicol, ciprofloxacin, and trimethoprim/sulfamethoxazole. The antibiotics were tested at concentrations 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 compound. A 1-mL aliquot of the culture was centrifuged at 10,000 rpm for 2 min. The cell pellet was resuspended in 500 [micro]L of sterile water and boiled for 10 min. The resulting DNA suspension was used as template DNA in polymerase 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 [micro]L of template DNA, 5 [micro]M 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 [micro]L. A "hot start" method was used, and 1.25 U of Taq DNA polymerase (Sigma, St. Louis, MO) was added after initial template denaturation. The PCR cycle was as follows: initial denaturation for 12 min at 94[degrees]C, hot start pause at 80[degrees]C followed by 35 cycles of denaturation at 94[degrees]C for 1 min, primer annealing at 60[degrees]C for 1 min, and extension at 72[degrees]C for 5 min at first cycle. An additional 5 s was progressively added to each cycle to reach a final of 7 min, 55 s. PCR products were analyzed on 1% agarose gel.

Amplification products were extracted from the gels with the QIAGEN QIAquick gel extraction kit (Valencia, CA). The amplified products were sequenced at a commercial facility (MWG Biotech Inc., High Point, NC) with the class 1 and class 2 integron variable region primers (integ and hep) (Table). Contiguous sequences were created from single sequence reads by using the CAP3 sequence assembly program (16). Contiguous sequences were analyzed by using the GenBank database of the National Center for Biotechnology Information and the BLASTX search engine (17). Putative gene relationships and sequence data were analyzed by using a multiple sequence alignment created by using Clustal W version 1.82 (18).