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. 2004 Oct;48(10):3996–4001. doi: 10.1128/AAC.48.10.3996-4001.2004

Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins

Yolanda Sáenz 1, Laura Briñas 1, Elena Domínguez 1, Joaquim Ruiz 2, Myriam Zarazaga 1, Jordi Vila 2, Carmen Torres 1,*
PMCID: PMC521888  PMID: 15388464

Abstract

Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E. coli strains, respectively.

The emergence of Escherichia coli isolates with multiple-antibiotic-resistant phenotypes, involving coresistance to four or more unrelated families of antibiotics, has been previously reported and is considered a serious health concern (2, 5, 22). Transference of resistance determinants by mobile genetic elements including plasmids, transposons, and gene cassettes in integrons (4, 13) and the alteration in mar locus regulation (1, 2, 27) are important factors that can contribute to the increase in multiresistant bacteria.

In previous studies (7, 34), the antibiotic resistance profiles of 515 nonpathogenic E. coli isolates obtained from food products of animal origin (n = 47) and from fecal samples of healthy animals (n = 177) and humans (n = 291) were studied. Seventeen E. coli isolates from those groups (four from food, eight from animals, and five from humans) showed a multiple-antibiotic-resistant phenotype (resistance to nalidixic acid, ampicillin, rifampin, chloramphenicol, sulfamethoxazole, streptomycin [STR] and tetracycline). All 17 of these isolates were used in the present work to detect different mechanisms of antibiotic resistance and to study the antibiotic resistance genes inside integrons and the relevance of the mar locus in the multiple-antibiotic-resistant phenotype.

Additional susceptibilities to ciprofloxacin, amoxicillin-clavulanic acid, cefazolin, cefoxitin, ceftazidime, cefotaxime, ceftriaxone, imipenem, aztreonam, gentamicin (GEN), apramycin, tobramycin, kanamycin, and trimethoprim were determined by an agar dilution method (24).

The 17 E. coli isolates showed 16 unrelated pulsed-field gel electrophoresis (PFGE) patterns with the XbaI enzyme in experiments following the method of Gautom (9) (Fig. 1). Only strains Co71 and Co82 showed closely related patterns.

FIG. 1.

FIG. 1.

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PFGE patterns of XbaI-digested genomic DNA of the 17 multiresistant E. coli strains. Lanes: 1, λ ladder molecular size marker; 2, E. coli Co1; 3, E. coli Co19; 4, E. coli Co45; 5, E. coli Co53; 6, E. coli Co71; 7, E. coli Co82; 8, E. coli Co80; 9, E. coli Co110; 10, E. coli Co125; 11, E. coli Co177; 12, E. coli Co201; 13, E. coli Co227; 14, E. coli Co228; 15, E. coli Co232; 16, E. coli Co279; 17, E. coli Co354; 18, E. coli Co356; 19, λ ladder molecular size marker.

Analysis of antibiotic resistance mechanisms.

The presence of antibiotic resistance genes in the 17 E. coli strains was analyzed by PCR, PCR-restriction fragment length polymorphism analysis, and sequencing (Table 1). Table 2 shows the resistance phenotypes and genes identified.

TABLE 1.

Primers and annealing temperatures used in the PCR reactions carried out in this study

Primer name Sequence (5′→3′) Target gene(s) or region PCR product size (bp) Annealing temp (°C) Reference
TEM-F ATTCTTGAAGACGAAAGGGC blaTEM 1,150 60 3
TEM-R ACGCTCAGTGGAACGAAAAC
SHV-F CACTCAAGGATGTATTGTG blaSHV 885 52 30
SHV-R TTAGCGTTGCCAGTGCTCG
OXA-F ACACAATACATATCAACTTCGC blaOXA 813 61 36
OXA-R AGTGTGTTTAGAATGGTGATC
AacC1-F ACCTACTCCCAACATCAGCC aac(3)-I 169 60 37
AacC1-R ATATAGATCTCACTACGCGC
AacC2-F ACTGTGATGGGATACGCGTC aac(3)-II 237 60 37
AacC2-R CTCCGTCAGCGTTTCAGCTA
AacC3-F CACAAGAACGTGGTCCGCTA aac(3)-III 185 60 37
AacC3-R AACAGGTAAGCATCCGCATC
AacC4-F CTTCAGGATGGCAAGTTGGT aac(3)-IV 286 60 37
AacC4-R TCATCTCGTTCTCCGCTCAT
Ant(2′′)-F ATGTTACGCAGCAGGGCAGTCG ant(2′′) 187 55 38
Ant(2′′)-R CGTCAGATCAATATCATCGTGC
AphA1-F ATGGGCTCGCGATAATGTC aphA1 600 50 22
AphA1-R CTCACCGAGGCAGTTCCAT
AphA2-F GAACAAGATGGATTGCACGC aphA2 680 50 22
AphA2-R GCTCTTCAGCAATATCACGG
AadA-F GCAGCGCAATGACATTCTTG aadA1 or aadA2 282 60 16
AadA-R ATCCTTCGGCGCGATTTTG 20
TetA-F GTAATTCTGAGCACTGTCGC tetA 937 62 11
TetA-R CTGCCTGGACAACATTGCTT
TetB-F CTCAGTATTCCAAGCCTTTG tetB 416 57 11
TetB-R CTAAGCACTTGTCTCCTGTT
TetC-F TCTAACAATGCGCTCATCGT tetC 570 62 11
TetC-R GGTTGAAGGCTCTCAAGGGC
TetD-F ATTACACTGCTGGACGCGAT tetD 1,104 57 11
TetD-R CTGATCAGCAGACAGATTGC
TetE-F GTGATGATGGCACTGGTCAT tetE 1,179 62 11
TetE-R CTCTGCTGTACATCGCTCTT
CmlA-F TGTCATTTACGGCATACTCG cmlA 455 55 This study
CmlA-R ATCAGGCATCCCATTCCCAT
FloR1 CACGTTGAGCCTCTATAT floR 868 55 26
FloR2 ATGCAGAAGTAGAACGCG 6
DfrIa-F GTGAAACTATCACTAATGG dfrA1, dfrA5, dfrA15, dfrA15b, dfrA16, dfrA16b 474 55 25
DfrIa-R TTAACCCTTTTGCCAGATTT
DfrIb-F GAGCAGCTICTITTIAAAGC dfrA14, dfrA6 393 60 25
DfrIb-R TTAGCCCTTTIICCAATTTT
DfrVII-F TTGAAAATTTCATTGATT dfrA7, dfrA17 474 55 25
DfrVII-R TTAGCCTTTTTTCCAAATCT
DfrII-F GATCACGTGCGCAAGAAATC dfrB1, dfrB2, dfrB3 141 60 25
DfrII-R AAGCGCAGCCACAGGATAAAT
DfrXII-F GGTGSGCAGAAGATTTTTCGC dfrA12, dfrA13 319 60 25
DfrXII-R TGGGAAGAAGGCGTCACCCTC
Sul-F TGGTGACGGTGTTCGGCATTC sul1 789 63 23
Sul-R GCGAGGGTTTCCGAGAAGGTG This study
Sul2-F CGGCATCGTCAACATAACC sul2 722 50 22
Sul2-R GTGTGCGGATGAAGTCAG
Sul3-F CATTCTAGAAAACAGTCGTAGTTCG sul3 990 51 29
Sul3-R CATCTGCAGCTAACCTAGGGCTTTGGA
IntI1-F GGGTCAAGGATCTGGATTTCG intI1 483 62 23
IntI1-R ACATGGGTGTAAATCATCGTC
IntI2-F CACGGATATGCGACAAAAAGGT intI2 788 62 23
IntI2-R GTAGCAAACGAGTGACGAAATG
Int-F GGCATCCAAGCAGCAAG Class 1 integron variable region Variable 55 19
Int-R AAGCAGACTTGACCTGA
Hep-F CGGGATCCCGGACGGCATGCACGATTTGTA Class 2 integron variable region Variable 60 39
Hep-R GATGCCATCGCAAGTACGAG
Qac-F GGCTGGCTTTTTCTTGTTATCG qacEΔ1 287 60 23
Qac-R TGAGCCCCATACCTACAAAGC
MarR-F AGCTAGCCTTGCATCGCA marR and marO 568 55 28
MarR-R TACGGCAGGACTTTCTTAAGCA
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TABLE 2.

Phenotypes and mechanisms of antibiotic resistance detected in the 17 multiresistant E. coli strains of this study

E. coli strain (origin)a Phenotype of resistanceb Mechanisms of resistance
Resistance genes detected Position(s) of amino acid change(s) in:
CATc
GyrA ParC
Co1 (F) Nal Cip Amp Kan Str Rif Tet Chl Tmp Smx blaTEM1b, aphA1, aphA2, aadA1, tetB, dfrA1, sul2 83 + 87 80
Co19 (F) Nal Amp Kan Str Rif Tet Chl Tmp Smx blaTEM1b, aphA2, aadA1, tetB, dfrA1, sul2 83
Co45 (F) Nal Cip Amp Amcd Kan Str Rif Tet Chl Tmp Smx blaTEM1b, aphA2, aadA1, tetA, dfrA1, sul2 83 + 87 80
Co53 (F) Nal Amp Amcd Kan Str Rif Tet Chl Smx blaTEM1b, aphA2, tetB, sul2 83
Co71 (B) Nal Cip Amp Kan Str Rif Tet Chl Tmp Smx blaTEM1b, aphA1, aadA5, tetB, dfrA17, sul1, sul2 83 + 87 80 +
Co80 (B) Nal Amp Gen Tobd Kan Str Rif Tet Chl Smx blaTEM1b, aac(3)-II, aphA2, aadA1, tetB, sul1, sul2 83 +
Co82 (B) Nal Cip Amp Kan Str Rif Tet Chl Tmp Smx blaTEM1b, aphA1, aadA5, tetB, dfrA17, sul1, sul2 83 + 87 80 +
Co110 (B) Nal Amp Amcd Str Rif Tet Chl Tmp Smx blaTEM1a, aadA1, tetA, cmlA, dfrA1, sul1, sul3 83
Co125 (P) Nal Cip Amp Str Rif Tet Chl Tmp Smx blaTEM1b, aadA1, aadA2, tetA, cmlA, dfrA12, dfrA1-like, sul3 83 + 87 80
Co279 (P) Nal Amp Amcd Str Rif Tet Chl Tmp Smx blaTEM1b, aadA1, aadA2, tetB, cmlA, dfrA12, sul3 83
Co177 (D) Nal Amp Amcd Str Rif Tet Chl Tmp Smx blaTEM1b, aadA1, tetA, dfrA1, sul1, sul2 83 +
Co201 (D) Nal Amp Amcd Str Rif Tet Chl Tmp Smx blaTEM1b, aadA1, tetA, dfrA1, sul1, sul2 83 +
Co227 (H) Nal Amp Amcd Gen Apr Tob Str Rif Tet Chl Tmp Smx blaTEM1b, aac(3)-IV, aadA1, aadA2, tetA, cmlA, dfrA1, dfrA12, sul1, sul2, sul3 83
Co228 (H) Nal Amp Amcd Gen Apr Tob Kan Str Rif Tet Chl Tmp Smx blaTEM1a, aac(3)-IV, aphA2, aadA2, tetA, cmlA, dfrA12, sul1, sul3 83
Co232 (H) Nal Cip Amp Str Rif Tet Chl Tmp Smx blaTEM1b, aadA1, tetA, dfrA1, sul1, sul2 83 + 87 80 + 84 +
Co354 (H) Nal Cip Amp Gen Apr Tob Str Rif Tet Chld Tmp Smx blaTEM1b, aac(3)-IV, aadA1, aadA2, tetA, dfrA1, dfrA12, sul1, sul3 83 + 87 80
Co356 (H) Nal Cip Amp Str Rif Tet Chl Tmp Smx blaTEM1b, aadA5, tetB, dfrA17, sul1, sul2 83 + 84 80 + 108 +
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a

F, food; B, broiler; P, pig; D, dog; H, human.

b

Nal, nalidixic acid; Cip, ciprofloxacin; Amp, ampicillin; Amc, amoxicillin-clavulanic acid; Gen, gentamicin; Apr, apramycin; Tob, tobramycin; Kan, kanamycin; Str, streptomycin; Rif, rifampin; Tet, tetracycline; Chl, chloramphenicol; Tmp, trimethoprim; Smx, sulfamethoxazole.

c

CAT, chloramphenicol-acetyl-transferase enzymatic activity. +, CAT was detected for the strain indicated.

d

Resistance to the drug indicated is in the intermediate category according to NCCLS standards for the corresponding strain.

All strains were ampicillin resistant, and for eight of them, the minimal inhibitory concentration (MIC) of amoxicillin-clavulanic acid indicated intermediate resistance; no strain was resistant to the remaining β-lactams studied. The blaTEM-1a and blaTEM-1b genes were identified in 2 and 15 strains, respectively, whereas none of the blaSHV and blaOXA genes were found.

Four strains in which the aac3-II (found in one strain from a broiler) or aac3-IV gene (found in three strains from humans) was found were GEN resistant. The AAC(3)-IV enzyme modifies apramycin in addition to GEN. Apramycin is used exclusively in veterinary medicine, but the GEN-related chemical structure and the mobility of the aac3-IV gene inside plasmids may have contributed to the selection and dissemination of these strains in a human environment (17).

Eight of the seventeen E. coli strains were kanamycin resistant, and the aphA1 and aphA2 genes were detected in three and six strains, respectively. Both genes were found in one strain of food origin.

The following aadA genes were detected in 16 of the 17 STR-resistant strains: aadA1 was found in 12 strains, aadA2 was found in 5, and aadA5 was found in 3 strains. The aadA1 and aadA2 genes were found together in four strains (two from pigs and two from humans). No STR resistance mechanism was detected in the Co53 strain, in which case other mechanisms of STR resistance, such as the production of APH(3′′)-I or APH(6)-I phosphoryltransferases (15, 35), cannot be excluded.

The tetA or tetB gene was found in all the strains (tetA was found in nine, and tetB was found in eight strains). No tetC, tetD, or tetE genes were detected. Chloramphenicol-acetyltransferase activity was demonstrated as previously described (8) in the seven strains for which the MICs of chloramphenicol were highest (≥128 μg/ml) (Table 2). The cmlA gene was detected in five additional strains (MICs of chloramphenicol, 32 to 64 μg/ml), while the floR gene was not found.

Fifteen E. coli strains were trimethoprim resistant, and the following dfr genes were identified by PCR-restriction fragment length polymorphism analysis (25): dfrA1 was found in seven strains, dfrA1 plus dfrA12 was found in two, dfrA1-like plus dfrA12 was found in one strain, dfrA17 was found in three, and dfrA12 was found in two strains. A new type of dfrA gene, called dfrA1-like, was found in the Co125 strain. Sequencing of the Co125 amplicon indicated a deduced amino acid substitution, Asn134Asp, in contrast to DHFRIa (Swiss-Prot accession number P00382).

The sul1 and sul2 genes were detected in 11 and 12 strains, respectively, and 8 of those strains showed both genes. These findings are in agreement with the high prevalence of these genes found in Enterobacteriaceae (18, 22). The sul3 gene has recently been found in pathogenic E. coli isolates (10, 12, 29), being detected in six of our strains (one from a broiler, two from pigs, and three from humans). The mechanisms of quinolone resistance had been previously analyzed in these 17 strains (7, 33).

Integron analysis.

Class 1 and class 2 integrons, the most frequently found in resistant bacteria (14, 23, 31, 32), were analyzed in all our strains. PCR amplification was used to detect class 1 and class 2 integrase genes, intI1 and intI2, respectively, and the qacEΔ1 gene. The variable regions (VR) of these integrons were studied by PCR and sequencing (Table 1). Twelve strains presented the intI1 gene and four presented the intI2 gene; one of these was E. coli Co125, which was positive for both genes.

The VR of the class 1 integron was analyzed in the 12 intI1-positive strains, and the following gene cassette arrangements were detected (Table 3): aadA1 (one strain), dfrA1 plus aadA1 (four strains), dfrA1 plus aadA1 and dfrA12 plus orfF plus aadA2 (two strains), dfrA12 plus orfF plus aadA2 (two strains), and dfrA17 plus aadA5 (three strains). Our E. coli strain Co125 gave unexpected results: the intI1 PCR was positive, whereas no qacEΔ1 or sul1 genes and no PCR amplicon of the class 1 integron VR were detected. Thus, Co125 was studied in detail by PCR mapping. A 1,650-bp amplicon was obtained using the primers Int-F and AadA-R. Sequencing revealed the presence of the dfrA12 plus orfF plus aadA2 gene cassettes (Table 3). As in the case of our results, most class 1 integrons published are composed of two or more gene cassettes (12, 22, 40, 41).

TABLE 3.

Analysis of class 1 and class 2 integrons and their resistance gene cassettes in the 17 multiresistant E. coli strains

E. coli strain (origin)a Class 1 integron
Class 2 integron
Detection ofb:
Variable region amplified by PCR
Detection of intI2 geneb Variable region amplified by PCR
intI1 qacEΔ1 sul1 Size of amplicons obtained (bp) Genes included in cassettes Size of amplicons obtained (bp) Genes included in cassettes
Co1 (F) − − NADc + 2,000 dfrA1 + sat + aadA1
Co19 (F) − − NAD + 2,000 dfrA1 + sat + aadA1
Co45 (F) − − NAD + 2,000 dfrA1 + sat + aadA1
Co53 (F) − − NAD − NAD
Co71 (B) + + 1,700 dfrA17 + aadA5 − NAD
Co80 (B) + + 1,000 aadA1 − NAD
Co82 (B) + + 1,700 dfrA17 + aadA5 − NAD
Co110 (B) + + 1,500 dfrA1 + aadA1 − NAD
Co125 (P) + − NAD dfrA12 + orfF + aadA2d + 2,000 dfrA1-like + sat + aadA1
Co279 (P) − − NAD − NAD
Co177 (D) + + 1,500 dfrA1 + aadA1 − NAD
Co201 (D) + + 1,500 dfrA1 + aadA1 − NAD
Co227 (H) + + 1,500, 2,000 dfrA1 + aadA1, dfrA12 + orfF + aadA2 − NAD
Co228 (H) + + 2,000 dfrA12 + orfF + aadA2 − NAD
Co232 (H) + + 1,500 dfrA1 + aadA1 − NAD
Co354 (H) + + 1,500, 2,000 dfrA1 + aadA1, dfrA12 + orfF + aadA2 − NAD
Co356 (H) + + 1,700 dfrA17 + aadA5 − NAD
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a

F, food; B, broiler; P, pig; D, dog; H, human.

b

+, detected; −, not detected.

c

NAD, No amplicon was detected.

d

These gene cassettes were detected by sequencing of amplicons obtained with the primers Int-F and AadA-R.

A class 2 integron carrying the dfrA1 plus sat plus aadA1 gene cassettes was detected in four strains (three from foods and one from a pig). The dfrA1 gene cassette detected in Co125 presented the Asn134Asp amino acid change, corresponding to the sequence of the dfrA1-like gene found in this strain (Table 3).

Analysis of the mar locus.

Another mechanism contributing to a multiple-antibiotic-resistant phenotype is associated with mar locus regulation (1, 2, 5). Amino acid changes in MarR and the nucleotide mutations in the operator-promoter region marO were studied for all strains by PCR, sequencing, and comparison with the GenBank sequence found under accession number M96235 and corresponding to the mar regulon (Table 4). Fifteen strains showed Gly103Ser and Tyr137His substitutions in MarR, which had been found also in resistant clinical strains (27). Note that position 103 is inside the conserved region (between amino acids 92 and 104 in MarR) and may be important for binding with the region corresponding to marO (1). However, other authors have considered that these substitutions could correspond to genotypic variations in marR without loss of repressor activity (27). Another amino acid change in MarR, Leu36Gln, was found in only one strain in our study; this is the first time that this substitution has been reported. Further studies are necessary to relate this substitution to antibiotic resistance.

TABLE 4.

Analysis of amino acid changes in MarR protein and nucleotide mutations in marO region of 17 multiresistant E. coli strainsa

E. coli strain(s) Amino acid changes in MarR Nucleotide mutation(s) in marO
Co53, Co356 None None
Co177 Gly103Ser, Tyr137His None
Co279 Gly103Ser, Tyr137His A1332C
Co1 Gly103Ser, Tyr137His A1332C, A1331G
Co110, Co232, Co354 Gly103Ser, Tyr137His A1332C, C1370T
Co201, Co227, Co228 Gly103Ser, Tyr137His A1332C, C1375T
Co71, Co82, Co125 Gly103Ser, Tyr137His A1332C, C1379T
Co19, Co80 Gly103Ser, Tyr137His A1332C, C1414T
Co45 Leu36Gln, Gly103Ser, Tyr137His A1332C
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a

The sequence found under GenBank accession number M96235 was used as a reference.

Regarding nucleotide mutations in marO, 14 strains showed the previously reported A1332C transversion (27) together with amino acid substitutions in MarR at positions 103 and 137. We identified other nucleotide mutations (at positions 1331, 1370, 1375, 1379, and 1414) not previously found in the literature. MarA is known to activate the marRAB operon binding the “marbox” region in marO, but the adjacent region (nucleotides 1329 to 1346) also plays a role in binding and increases this transcriptional activation (21). Indeed, the mutations found at positions 1331 and 1332 are located inside this adjacent region, but additional studies should determine their effect on MarA activity. We found no differences in resistance phenotype between the strains with and without these mutations.

Our results show a wide variety of resistance genes in multiresistant nonpathogenic E. coli strains from humans, animals, and food products. Therefore, this normal flora may play a key role as an acceptor and donor of transmissible antimicrobial resistance mechanisms. The inclusion of some resistance genes inside integrons constitutes an effective means to spread antibiotic resistance among bacteria from different ecosystems. Moreover, different amino acid changes in MarR and mutations in marO were found, possibly contributing to the multiple antibiotic resistance phenotype.

Acknowledgments

This work has been partially supported by grants from the Fondo de Investigaciones Sanitarias (FIS 01/973) and the Consejería de Educación del Gobierno de La Rioja (ACPI2001/04) of Spain. Y. Sáenz has a fellowship from the University of La Rioja (FPI-UR-00/72785864).

We are grateful to R. Del Campo for critical review of the manuscript.

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