RESEARCH Table. Oligonucleotide primer sequences used for amplification of class 1 and class 2 integrase and variable regions Primer Primer sequence Target Reference integ-1 5´-GGCATCCAAGCAAG-3´ 5′-Class 1 integron variable region Levesque et al. 1995 (13) integ-2 5´-AAGCAGACTTGACCTGA-3´ 3´-Class 1 integron variable region Levesque et al. 1995 (13) hep51 5´-GATGCCATCGCAAGTACGAG-3´ 5´-Class 2 integron variable region White et al. 2001 (14) hep74 5´-CGGGATCCCGGACGGCATGCACGATTTGTA-3′ 3´-Class 2 integron variable region White et al. 2001 (14) intI1.F 5´-GGGTCAAGGATCTGGATTTCG-3´ 5´-intI1 gene Mazel et al. 2000 (15) intI1.R 5´-ACATGGGTGTAAATCATCGTC-3´ 3´-intI1 gene Mazel et.al. 2000 (15) intI2.F 5´-CACGGATATGCGACAAAAAGGT-3´ 5´-intI2 gene Mazel et al. 2000 (15) intI2.R 5´-GTAGCAAACGAGTGACGAAATG-3´ 5´-intI2 gene Mazel et al. 2000 (15) added after initial template denaturation. The PCR cycle was as follows: initial denaturation for 12 min at 94°C, hot start pause at 80°C followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and extension at 72°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 commer- cial 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 assem- bly 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 cre- ated by using Clustal W version 1.82 (18). Results Of the 322 E. coli isolates from sediment and irrigation water samples analyzed for antimicrobial resistance, 104 (32%) isolates showed resistance to at least one of the antimicrobial compounds (Figure 1). Approximately 10% (32/322) of all the isolates showed a multidrug resistance phenotype. Eighteen percent of the isolates were resistant to cephalothin; however, only 5 (2%) of 322 were resistant to ceftriaxone, which also belongs to the cephalosporin family. Resistance to ampicillin was prevalent in approxi- mately 35 (11%) of the isolates. Resistance to tetracycline (9%), kanamycin (2%), gentamicin (0.3%), and strepto- mycin (4%) was also observed. Resistance to the fluoro- quinolone ciprofloxacin was seen in one isolate. Three (<1%) of the 322 isolates were resistant to sulfonamide sulfamethoxazole. On the basis of analysis of variance, antimicrobial resistance and the sampling location were correlated. Isolates from the El Paso sampling region had significantly higher (p<0.05) antimicrobial resistance as compared with the Presidio and Weslaco sampling regions (data not shown). The 32 isolates identified as multiple antimicrobial resistant were assayed by PCR amplification for class 1 and class 2 integrase genes. Four isolates (approximately 13%) had the class 1 integrase gene intI1 (Figure 2A), and one isolate had the class 2 integrase gene intI2 (Figure 2B). Isolates identified as having the class 1 or class 2 integrase genes were further characterized through PCR amplifica- tion of the class 1 and class 2 variable regions. Of the four amplified class 1 integron variable regions, three isolates (isolate 16, isolate 19, and isolate 21) were approximately 1 kb in size, but the fourth isolate (isolate I-6) harbored a 2-kb fragment (Figure 3A). The 1-kb amplification prod- ucts were observed in isolates from the El Paso area. Nucleotide sequencing showed that all of the 1-kb sequences contained a conserved configuration of a 780- bp gene cassette identified as the aadA gene (Figure 4). The 2-kb amplification product was seen in an isolate from the Presidio sampling region. Nucleotide sequencing showed that the variable region contained a 498-bp gene cassette, identified as the dhfrXII gene, which encodes trimethoprim resistance. The gene cassette did not exhibit perfect homology with the dhfrXII gene (Figure 4). Within the identified 498-bp gene cassette, a 323-bp stretch showed 97% sequence homology; in addition, 59-bp and 56-bp fragments showed 88% and 89% homology, respec- tively. “Islands” of sequence within the variable region showed no sequence homology to any known genes. When the 32 multiply antimicrobial-resistant isolates were screened for class 2 integrons, only 1 isolate was pos- Figure 1. Frequency of antimicrobial resistance observed in 322 Escherichia coli isolates from irrigation water along the Texas- Mexico border. Emerging Infectious Diseases • Vol. 9, No. 7, July 2003 823