Article Figures & Data
Figures
- FIG. 1.
OMP profiles of selected E. coli isolates and control strains grown in nutrient broth. Proteins were separated on 10% polyacrylamide-8 M urea gels and stained with Coomassie blue. (A) Comparison of isolates with phenotypes designated as A (EC3589, EC3437, EC922, and EC3095) or B (EC689, EC3194, EC3076) and control strains E. coli C600 (wild type), E. coli MH225 (OmpC−), and CDC3100 (enhanced AmpC producer). (B) Comparison of isolates with phenotypes designated as C (EC251), D (EC4081), A (EC3006), or E (EC1552) and E. coli C600 and E. coli MH225 (OmpC−).
- FIG. 2.
DNA sequence of the ampC promoter and attenuator regions from selected E. coli isolates compared with analogous sequences of K12 and DH5α (4, 5). The −35 and −10 consensus promoter sequences and the translation initiation codon for AmpC β-lactamase are underlined. Arrows indicate the transcription attenuator region. The amino acid sequence of the initial segment of the AmpC leader region is shown below the corresponding nucleotide sequence.
Tables
- TABLE 1.
Primers used for PCR amplification of β-lactamase genes
Gene Primer Sequence (5′→3′) PCR product size (pb) E. coli ampC promoter AB1 GATCGTTCTGCCGCTGTG 271 AmpC2 GGGCAGCAAATGTGGAGCAA TEM Forward ATGAGTATTCAACATTTCCG 867 Reverse CTGACAGTTACCAATGCTCTC SHV Forward GGTTATGCGTTATATTCGCC 867 Reverse TTAGCGTTGCCAGTGCTC OXA-1 OXA-1F ACACAATACATATCAACTTCGC 814 OXA-1R AGTGTGTGTTTAGAATGGTGATC OXA-2 OXA-2F TTCAAGCCAAAGGCACGATAG 704 OXA-2R TCCGAGTTGACTGCCGGGTTG OXA-10 OXA-10F CGTGCTTTGTAAAAGTAGCAG 651 OXA-10R CATGATTTTGGT GGGAATGG - TABLE 2.
β-Lactam resistance phenotypes for 59 E. coli isolates for which the MIC of cefpodoxime is ≥2 and that of ceftazidime is ≤1 μg/mla
MIC (μg/ml) Major phenotypes Minor phenotypes A B C D E n strains (%) 32 (54.2%) 18 (30.5%) 7 (11.9%) 1 (1.7%) 1 (1.7%) β-Lactamase pI 5.4 ≥8.5 7.2 5.4, 5.7, 6.8 AMX >64 32->64 >64 4 >64 AMX-CLAV 16-32 16-32 32->64 4 32 TIC >128 8-32 >128 8 >128 PIP >128 <4-8 >128 16 >128 PIP-TZ 8->128 <4-8 ≥128 16 16 CFZ 16->256 4-64 16-32 2 16 CFZ-CLAV 2-4 2-16 4-8 1 2 CXM 16-32 8-32 32-128 16 128 CPD 2-4 2-4 4-8 4 2 FOX 16->32 8-32 8-32 16 16 CAZ 0.5-1 0.5-1 0.25-1 0.5 1 CAZ-CLAV 0.5 0.5-1 0.12-0.5 0.5 0.5 CTX 0.5 0.25-1 1 1 0.5 CTX-CLAV 0.25-0.5 0.12-0.5 0.25 0.5 0.5 FEP 0.12-0.5 0.06-0.25 2-4 0.25 0.5 FEP-CLAV 0.12 0.06-0.12 0.12-0.5 0.12 0.12 ↵a NCCLS breakpoints (S/I/R): AMX, CAZ, CXM, CFZ, FEP, FOX, ≤8/16/≥32; CPD, ≤2/4/≥8; CTX, ≤8/16-32/≥64; PIP, TIC, ≤16/32-64/≥128
- TABLE 3.
MICs for EC410 and EC3437 and transconjugants
Antibiotic MIC (μg/ml) for: HB101 EC410 HB101/410TC EC3437 HB101/3437TC AMX 4 >64 >64 >64 >64 AMX-CLAV 4 16 8 16 4 TIC ≤4 >128 >128 >128 >128 PIP ≤4 >128 >128 >128 >128 PIP-TZ ≤4 ≤4 ≤4 32 ≤4 CFZ 2 16 4 32 4 CFZ-CLAV 1 4 1 4 1 CXM 4 32 4 16 4 CXM-CLAV 2 16 2 16 2 FOX 4 32 4 32 4 CPD 1 2 1 4 1 CPD-CLAV 0.5 2 0.5 2 0.5 CAZ 0.5 0.5 0.5 0.5 0.5 CAZ-CLAV 0.25 0.25 0.25 0.5 0.25 CTX 0.12 0.5 0.12 0.25 0.12 CTX-CLAV 0.12 0.25 0.06 0.25 0.12 ATM 0.25 0.5 0.25 0.5 0.25 ATM-CLAV 0.12 0.5 0.12 0.25 0.25 FEP 0.06 0.12 0.06 0.12 0.12 FEP-CLAV 0.06 0.06 0.06 0.12 0.06 a Strains EC410, EC3437 and their respective transconjugants harbored a pI 5.4 TEM beta-lactamase (see characterization of phenotype A).
- TABLE 4.
Resistance profiles of EC251 and EC3006 and their respective transconjugants and clonesa
Antibiotic MIC (μg/ml) HB101 DH5α EC251 HB101 251TC DH5α (pBG251) EC3006 HB101 3006TC DH5α(pBG3006) AMX 4 ≤2 >64 >64 >64 >64 >64 >64 AMX-CLAV 4 ≤2 32 16 32 32 16 32 TIC ≤4 ≤4 >128 >128 >128 >128 >128 >128 PIP ≤4 ≤4 >128 64 >128 >128 >128 >128 PIP-TZ ≤4 ≤4 128 16 >128 >128 128 >128 CZ 2 1 16 4 32 >256 32 256 CZ-CLAV 1 1 4 1 1 2 1 1 CXM 4 2 32 8 64 32 8 16 CXM-CLAV 2 1 16 4 4 16 4 2 FOX 4 2 32 4 2 16 4 2 CPD 1 0.5 4 1 16 4 1 2 CPD-CLAV 0.5 0.5 2 0.5 0.5 2 0.5 0.25 CAZ 0.5 0.25 0.5 0.25 0.25 1 0.5 0.5 CAZ-CLAV 0.25 0.25 0.25 0.25 0.12 0.5 0.25 0.25 CTX 0.12 0.06 1 0.5 4 0.5 0.12 0.12 CTX-CLAV 0.12 0.06 0.25 0.12 0.12 0.5 0.06 0.06 ATM 0.25 0.06 0.5 0.12 0.5 0.5 0.25 0.12 ATM-CLAV 0.12 0.06 0.5 0.12 0.06 0.25 0.12 0.12 FEP 0.06 0.06 2 1 >32 0.5 0.12 0.5 FEP-CLAV 0.06 0.03 0.12 0.12 0.25 0.12 0.06 0.12 ↵a EC251 and its corresponding transconjugant (HB101 251TC) and clone [DH5α (pBG251)] harbored an OXA-30 β-lactamase (see characterization of phenotype C) and EC3006 and its corresponding transconjugant (HB101 3006TC) and clone [DH5α (pBG3006)] harbored a TEM-1 β-lactamase.
- TABLE 5.
Mutations in ampC promoter and attenuator regions and cephalosporinase specific activity of selected E. coli isolates
Strain Mutations in promoter and attenuatora Cephalo- sporinase sp actb DH5α Same as E. coli K-12 control 1.00 EC689 −73, −66, −28, +17 2.59 ± 0.26 EC3076 −76, +22, +26, +27, +32 2.04 ± 0.13 EC3194 −76, +22, +26, +27, +32 2.67 ± 0.12 EC3358 No mutations detected 2.40 ± 0.09 Control strains CDC10 −73, −28, 1-bp insert between −35 and −10, +17 13.6 ± 2.2 CDC110 −73, −32, +58 14.6 ± 1.3 CDC3100 −76, 2-bp insert between −35 and −10, +22, +26, +27, +32 17.2 ± 3.7
