Haemophilus influenzae bla _ROB-1 Mutations in Hypermutagenic ΔampC Escherichia coli Conferring Resistance to Cefotaxime and β-Lactamase Inhibitors and Increased Susceptibility to Cefaclor

Bacterial strains and GB20 construction. E. coli MI1443 (11) was used as the recipient for plasmids and for the determination of antibiotic susceptibilities. Phage P1 was grown on hypermutable E. coli strain AB1157 mutS::Tn10 with a defective mutS gene (32). Recovered phage P1 was then used to transduce E. coli MI1443 with a defective ampC gene, giving rise to E. coli GB20. Detailed descriptions of the strains are provided in Table 1.

Plasmids.Plasmid pMON401 (Amp^r Chl^r) harboring the bla_ROB-1 gene (20) is a pACYC184 (Tet^r Chl^r) derivative (9). Plasmids pMON410, pMON411, pMON420, pMON421, and pMON422 are pMON401 derivatives obtained in different serial passage experiments in this work. Plasmid pBGS18⁻ (Kan^r) (39) was used in subcloning experiments, giving rise to recombinant plasmids pBB1 (wild-type bla_ROB-1 gene); pBB10 and pBB11, which carry mutations in the bla_ROB-1 gene that confer resistance to β-lactam plus β-lactamase inhibitor combinations; as well as pBB20, pBB21, and pBB22, which have mutations that lead to increased cefotaxime-hydrolyzing activity. Plasmid pBB120 harbors the bla_ROB-1 gene with the changes that confer both clavulanate and cefotaxime resistance. Detailed descriptions of the plasmids are included in Table 1.

Determination of mutation frequencies.To ascertain the hypermutable nature of the E. coli GB20 construction, 50 μl of a 10⁻⁴ dilution of overnight cultures of each of the strains MI1443, GB20 (with and without pMON401), and AB1157 mutS::Tn10 was inoculated into five independent flasks containing Luria-Bertani (LB) broth; and the flasks were incubated overnight at 37°C under high agitation. A total of 100 μl of each overnight culture was plated onto LB agar plates containing 100 μg of rifampin per ml or 40 μg of nalidixic acid per ml. Simultaneously, 100 μl of a 10⁻⁶ dilution of each of the same cultures was plated on LB agar plates without antibiotics. The number of rifampin-resistant or nalidixic acid-resistant mutants was counted at 48 h, and the number of viable cells was counted at 24 h. The mutation frequency was defined as the ratio of rifampin- or nalidixic acid-resistant colonies versus the number of viable cells in antibiotic-free medium.

Serial passage experiments.Aliquots (5 μl) of an overnight culture of E. coli GB20(pMON401) were inoculated into five tubes containing 5 ml of LB broth with tetracycline (20 μg/ml) and chloramphenicol (30 μg/ml) to ensure the maintenance of Tn10 and pMON401, respectively. The cultures were grown at 37°C under normal agitation and were submitted to daily serial passages with increasing concentrations of cefotaxime alone or amoxicillin plus a fixed concentration of a β-lactamase inhibitor (2 μg of clavulanate per ml). The β-lactam concentrations ranged from 2 dilutions below to 6 dilutions above the MIC for GB20(pMON401). Plasmid DNA was extracted from each of the five bacterial populations that grew at 4 to 5 and 6 dilutions above the MIC of cefotaxime or 4 to 5 dilutions above the MIC of amoxicillin-clavulanate. DNA was introduced into MI1443 by transformation; transformants were selected on LB agar plates containing chloramphenicol (30 μg/ml) and cefotaxime or amoxicillin-clavulanate at 2, 3, or 4 dilutions above the MIC. Ten transformants from each population were chosen; and different phenotypes were identified by disk diffusion tests with cephalothin, cefazolin, cefuroxime, cefotaxime, ceftazidime, ampicillin, and amoxicillin-clavulanate. The plasmid DNA of transformants corresponding to each phenotype was reintroduced by transformation into MI1443, with only chloramphenicol used as a selector. The DNA of derivatives of strain MI1443(pMON401) with possible bla_ROB-1 gene variants was sequenced. Finally, each bla_ROB-1 gene variant was subcloned into plasmid pBGS18⁻ to ascertain the specificities of the phenotypic changes independently from the host plasmid.

Construction of plasmids pBB1, pBB10, pBB20, and pBB120.To avoid the possible interference of putative mutations in the bacterial resistance phenotype that could arise in other sites of plasmid pMON401 during serial passage experiments, the bla_ROB-1 gene was amplified from plasmids pMON401, pMON410, pMON411, pMON420, pMON421, and pMON422 by PCR and subcloned into pBGS18⁻, giving rise to recombinant plasmids pBB1, pBB10, pBB11, pBB20, pBB21, and pBB22, respectively. Plasmid pBB120 was constructed as described below.

Susceptibility testing.The MICs of amoxicillin, ampicillin, piperacillin, amoxicillin-clavulanate (2:1 ratio), ampicillin-sulbactam (2:1 ratio), piperacillin-tazobactam (fixed concentration of tazobactam, 4 μg/ml), cephalothin, cefuroxime, cefaclor, cefotaxime, ceftazidime, and imipenem were determined as recommended by the NCCLS (27).

Amplification and sequencing procedures.The primers used to amplify the bla_ROB-1 gene were ROBFE (5′-GGGGAATTCGATCAGAGTAATAATTTCTGAT-3′), which recognizes a region immediately upstream of the −35 region of the putative promoter, and ROBRH (5′-CGGCAAGCTTGAAAGCAAGTTTCAACGGCTT-3′), which hybridizes to a region 42 bp downstream of the stop codon containing an EcoRI and a HindIII restriction site (the respective restriction sites are underlined). The internal primers used to sequence the gene in both directions were ROBd1 (5′-CGTTTATGTATGGGATACAGAA-3′), which hybridizes to a region 185 bp from the beginning of the gene, and ROBr (5′-GGGTTACGTTATCGCCTAATT-3′), which hybridizes to a region 390 bp from the end of the gene. Amplification was performed in a mixture containing (per 50 μl) 2 mM MgCl₂, 1× PCR buffer II (Applied Biosystems, Weiterstadt, Germany), 200 μM (each) deoxynucleoside triphosphates, 25 pmol of each primer, and 2.5 U of Taq-Gold DNA polymerase (Applied Biosystems). The following PCR program was used: 94°C for 12 min and 35 amplification cycles of denaturation at 94°C for 60 s, annealing at 56°C for 60 s, and elongation at 72°C for 60 s. The amplification product was analyzed by electrophoresis on a 0.8% agarose gel predyed with ethidium bromide. The primers were removed from the PCR product with the QIAquick PCR purification kit (Qiagen, GmbH, Hilden, Germany) before the product was sequenced or cloned into a new plasmid.

Construction of β-lactamases with triple mutations.The enzyme with the triple mutation Ser130Gly, Arg164Trp, and Ala237Thr was constructed by using the new primers ROBArg164Trp (5′-CTAGCCA^∗ATTGGTATGGGTT-3′), which harbors the mutation G⁵³²→A⁵³² (asterisk) and which results in the Arg164Trp substitution, and ROBd2 (5′-TTGGCAAAGGCATGACGATTG-3′), which hybridizes to nucleotide positions 380 to 400 in the bla_ROB-1 gene, together with primers ROBFE and ROBRH described above. A first PCR was performed with primers ROBFE and ROBArg164Trp by using DNA from the amoxicillin-clavulanate-resistant mutant as the template. A second PCR was performed with primers ROBd2 and ROBRH by using DNA from the cefotaxime-resistant mutant. A third PCR was carried out with primers ROBFE and ROBRH by using the PCR products obtained in the previous amplifications as the templates. The final amplification product was digested with EcoRI and HindIII, ligated to pBGS18−, digested with the same restriction enzymes, and transformed into MI1443. Transformants were selected on LB agar plates containing kanamycin (50 μg/ml), and the bla_ROB gene of recombinant plasmid pBB120 was sequenced to confirm the presence of the three desired changes.

Enzyme kinetics.The β-lactamases extracted from E. coli MI1443(pBB1), MI1443(pBB10), MI1443(pBB11), MI1443(pBB20), MI1443(pBB21), and MI1443(pBB22) were prepared from 1 liter of an overnight culture of LB broth with kanamycin (50 μg/ml) at 37°C. The cells were harvested by centrifugation at 4,500 × g for 20 min, washed twice with 5 ml of 0.05 M phosphate buffer (pH 7.4), and resuspended in 2 ml of the same buffer. The suspension was sonicated for 10 min with 2-s pulses (Sonicator Heat system; Ultrasonics Inc.). Debris was removed by centrifugation (17,500 × g for 30 min at 4°C), and the β-lactamase activity of the supernatant was assayed by nitrocefin hydrolysis.

The β-lactamase activities of the extracts against different β-lactam antibiotics were measured spectrophotometrically (Uvikon-940 spectrophotometer). K_m and V_max values were obtained by double-reciprocal (Lineweaver-Burk) plots of the initial steady-state rates at different substrate concentrations. The inhibitor concentrations required to inhibit 50% of the β-lactamase activity (IC₅₀s) were determined by incubating the β-lactamase extracts with different concentrations of the inhibitors for 10 min at room temperature, and then 100 μM nitrocefin was added as the substrate (5).

Mutation frequency of constructed strain E. coli GB20.The mutation frequency of the constructed variant, E. coli GB20 (ΔampC, mutS defective), was compared with those of its isogenic strain, E. coli MI1443 (ΔampC), and strain E. coli AB1157 mutS::Tn10. The frequencies of mutation to rifampin (100 μg/ml) and nalidixic acid (40 μg/ml) resistance for E. coli GB20 were 2.7 ± 0.9 × 10⁻⁷ and 5.5 ± 3.2 × 10⁻⁷, respectively. Identical values were also obtained when the strain harbored plasmid pMON401 (data not shown). These mutation frequency values are similar to those for E. coli AB1157 mutS::Tn10 strain from which the mutS allele was derived, which were 4.5 ± 1.4 × 10⁻⁷ and 2.6 ± 0.5 × 10⁻⁷, respectively. For wild-type strain E. coli MI1443, the mutation frequencies were 2.6 ± 3.3 × 10⁻⁹ and 5.7 ± 2.9 × 10⁻⁹, respectively. These results showed that the mutation frequency for E. coli GB20 was 100-fold higher than that for its isogenic strain, E. coli MI1443. These results, which reflect the means of five replicate experiments in each case, coincided with those expected for hypermutable strains with a defective mutS gene (25).

Serial passage experiments.The original cefotaxime MIC for E. coli GB20(pMON401) was 0.06 μg/ml. After a week of daily passages with increasing cefotaxime concentrations, the strain was able to grow in the presence of cefotaxime at a concentration of up to 4 μg/ml. Transformants obtained from populations growing in five different tubes containing 2 or 4 μg of cefotaxime per ml exhibited a single phenotypic pattern of antibiotic resistance. Conversely, transformants from populations recovered from tubes containing only 1 μg of cefotaxime per ml yielded two different phenotypic patterns. This observation suggests that different cefotaxime-resistant mutational variants were selected in the presence of low antibiotic concentrations but were replaced by a more effective mutant in the presence of higher concentrations.

The original amoxicillin-clavulanate MIC for E. coli GB20 was 4/2 μg/ml, and after serial passages, growth was obtained in the presence of 64/2 μg/ml. Transformants obtained from all tubes containing this highly resistant population had a single phenotypic pattern of resistance. As in the case of cefotaxime, when transformants were obtained from cultures growing in tubes with only 16/2 μg of amoxicillin-clavulanate per ml, three different phenotypic patterns of antibiotic resistance were found. Again, the interpretation is that mutational variants were selected at low amoxicillin-clavulanate concentrations; however, only the more effective mutant (that with the “winner” genotype) was able to survive in the presence of the higher concentration.

Genetic characterization of mutants.Both strands of the bla_ROB-1 gene from E. coli strains that achieved the highest levels of amoxicillin-clavulanate or cefotaxime resistance after antibiotic challenge were fully sequenced. The mutations found are shown in Table 2.

Three types of mutant variants were obtained in the cefotaxime challenge experiments. Mutant ES1 was the only transformant found in all five tubes with the highest cefotaxime concentration. This variant showed two nucleotide substitutions, C436→T436 and G656→A656, resulting in the amino acid substitutions Arg164Trp (near the Ω loop) and Ala237Thr (next to the KSG motif, according to the numbering scheme of Ambler et al. [1]). Other extended-spectrum ROB (ES-ROB) variants were found only in the presence of lower cefotaxime concentrations. One of the mutants had the single mutation C436→T436, which gave rise to the Arg164Trp substitution; and the other mutant had the single change T452→C452, which resulted in the amino acid substitution Leu169Ser.

Two types of resistant mutants were detected in the amoxicillin-clavulanate challenge experiments. In the case of transformants from five of five tubes growing in the presence of high amoxicillin-clavulanate concentrations, a single type of inhibitor-resistant ROB (IR-ROB) mutant was detected. The mutant had the single mutation A335→G335, which results in the amino acid change Ser130Gly, located in the SDN motif of the β-lactamase molecule. A second type of IR-ROB mutant was detectable only among transformants growing in some tubes with low amoxicillin-clavulanate concentrations. That mutant had a single mutation, C674→T674, that conferred the amino acid change Arg244Cys.

Antibiotic susceptibilities of E. coli MI1443 containing pBB1 pBB10, pBB11, pBB20, pBB21, pBB22, and pBB120 recombinant plasmids.The MICs of the different β-lactam antibiotics for E. coli MI1443 harboring plasmid pBGS18⁻ and the recombinant plasmids (pBGS18⁻ derivatives with the bla_ROB variants) are shown in Table 3.

The E. coli strain with pBB20, which harbors the ES-ROB mutant enzyme with the substitutions Arg164Trp and Ala237Thr (the winner genotype in cefotaxime challenge experiments), had increased levels of resistance to extended-spectrum cephalosporins, ranging from 16-fold increased levels of resistance to ceftazidime to about 60-fold increased levels of resistance to cefotaxime, the selector antibiotic. However, this mutant was more susceptible to penicillins and, surprisingly, to cefaclor (16-fold) than the E. coli strain harboring the wild-type enzyme. The E. coli strain with pBB21, which carries Arg164Trp as the sole change, was 16 times more resistant to ceftazidime and cefotaxime than the E. coli strain harboring the wild-type enzyme; and the level of resistance of the E. coli strain with pBB21 to cefaclor was reduced 256-fold. For the E. coli strain with pBB22 (Leu169Ser), the ceftazidime MIC increased eightfold and the cefotaxime MIC increased fourfold, while the cefaclor MIC decreased eightfold.

The E. coli strain with pBB10, which harbors the IR-ROB enzyme with the Ser130Gly replacement (the winner genotype in amoxicillin-clavulanate challenge experiments), was eightfold more resistant to β-lactam-β-lactamase inhibitor combinations than the E. coli strain with pBB1 and the wild-type enzyme. Interestingly, the susceptibilities to all cephalosporins tested increased, particularly that to cefaclor (≥1,024-fold). In the case of the E. coli strain with pBB11, which harbors the second IR-ROB variant with the Arg244Cys amino acid change, the level of resistance to the inhibitors increased 4-fold, but again, the level of resistance to cefaclor decreased dramatically (≥1,024-fold).

The E. coli strain with the hybrid mutant enzyme (IR-ES-ROB) showed a phenotype of reduced levels of resistance to both amoxicillin-clavulanate (8 μg/ml, versus 4 μg/ml for the wild type) and ceftazidime (32 μg/ml, versus 8 μg/ml for the wild type). Other investigators have observed this trend with other β-lactamases (34, 40), once more revealing the difficulty in obtaining both resistance phenotypes in the same enzyme molecule.

Kinetic parameters.The kinetics of substrate hydrolysis by the ROB-1 β-lactamase in comparison with those by the ROB-1 mutants with a single mutation (IR-ROB or ES-ROB) or double mutations (mutant ES1) are shown in Table 4. With respect to cefotaxime, the catalytic efficiencies (V_max/K_m values) of the β-lactamases of the ES-ROB mutants with the single Leu169Ser or Arg164Trp change or the double Ala237Thr and Arg164Trp changes were 3.5, 5.5, and 1,000 times higher, respectively, than that of the ROB-1 β-lactamase. On the contrary, the catalytic efficiency toward cefaclor in these mutants was decreased 3, 17, and 6 times, respectively. The catalytic efficiency of the β-lactamase of the more efficient mutant (with double Ala237Thr and Arg164Trp mutations) toward ampicillin was reduced 27.5-fold.

The catalytic efficiencies (V_max/K_m values) of the IR-ROB β-lactamases with the Ser130Gly and the Arg244Cys changes toward cefaclor were reduced 23 and 95 times, respectively, in comparison with that of the wild-type enzyme. The catalytic efficiency toward ampicillin was also reduced 6.5-fold.

Activities of β-lactam inhibitors against ROB-1 β-lactamase variants.The IC₅₀s of clavulanate, sulbactam, and tazobactam for the wild-type ROB-1 β-lactamase and the different ROB-1 variants are shown in Table 5. The IC₅₀s of tazobactam for the variants compared with that for the wild-type enzyme increased the least: 70-fold for the ROB-1 variant carrying the Ser130Gly change and only 1.5-fold for the mutant with the Arg244Cys substitution. The IC₅₀s of clavulanate, sulbactam, and tazobactam increased the least for the ES-ROB mutant with the double Arg164Trp and Ala237Thr mutations, with the IC₅₀s being 30, 8.5, and 60 times lower, respectively, than the IC₅₀ for ROB-1.