Role of Permeability in the Activities of β-Lactams against Gram-Negative Bacteria Which Produce a Group 3 β-Lactamase

Abstract

The production of a group 3 β-lactamase permitted Escherichia coli to raise the MICs of ceftazidime, cefpirome, and meropenem greatly but those of imipenem and piperacillin only slightly. The ratios of maximum rate of hydrolysis to K_m of ceftazidime, cefpirome, and piperacillin were lower than those of meropenem and imipenem for the group 3 β-lactamase. The permeability coefficients for piperacillin and meropenem were higher than those for ceftazidime and cefpirome. Imipenem had the highest permeability coefficient.

The group 3 β-lactamases are metalloenzymes that hydrolyze carbapenems in addition to cephalosporins and penicillins (1). Recently, pathogens, such as Pseudomonas aeruginosa (7, 13) and Serratia marcescens (9), with group 3 β-lactamases encoded by plasmids have been isolated from clinical specimens collected in Japan. P. aeruginosa S-860/pMS381, which produces a group 3 β-lactamase and a moderate quantity of D2 porin, was found to be highly resistant to ceftazidime and cefpirome, intermediate to imipenem, and susceptible to piperacillin, even though all these antibiotics were hydrolyzed by this enzyme at a readily measurable rate (7). P. aeruginosa S-861/pMS382, which has the same enzyme but a reduced amount of D2 porin, was found to be highly resistant to imipenem in addition to ceftazidime and cefpirome and susceptible to piperacillin (7). These results suggest that not only the hydrolysis by β-lactamase but also the outer membrane barrier may play an important role in the resistance to β-lactams in the group 3 β-lactamase producers.

In this study, a plasmid encoding a group 3 β-lactamase was introduced into Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, and Klebsiella oxytoca. When analyzed by susceptibility testing, β-lactams showed various degrees of the reduction in activities against transformant strains in comparison with their parent strains. Therefore, using E. coli as a representative gram-negative bacteria, we evaluated the role of outer membrane permeability in the activities of some β-lactams against the group 3 β-lactamase producers.

S. marcescens W-313/pSW313 was isolated from a clinical specimen. The following strains, which are susceptible to various antibiotics, were used as recipient strains for transformations (4): E. coli MC4100 (3), C. freundii GN16922, K. pneumoniae GN19409, and K. oxytoca GN19428. These strains do not produce chromosomal β-lactamase constitutively. Plasmid pHSG398 was used as the cloning vector (11).

Plasmid DNA was prepared by the rapid alkaline extraction method (5). Restriction endonuclease (EcoRI) and T4 DNA ligase were purchased from Nippon Gene Co. Ltd., Toyama, Japan. Plasmid pSW313, encoding a group 3 β-lactamase, was introduced into E. coli MC4100 by electroporation (5) and into C. freundii GN16922, K. pneumoniae GN19409, and K. oxytoca GN19428 by the calcium chloride method (5).

Antibiotics were obtained from the following sources: cephaloridine, Sigma Chemical Co., St. Louis, Mo.; ceftazidime, Japan Glaxo Co., Tokyo, Japan; cefpirome, Shionogi Co., Ltd., Osaka, Japan; imipenem, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; meropenem, Sumitomo Pharmaceuticals Ltd., Osaka, Japan; and piperacillin, Toyama Chemical Co., Ltd., Tokyo, Japan. MICs were determined by the agar dilution method (7).

The group 3 β-lactamase was purified from E. coli MC4100/pSW313 according to the method described previously (7). Molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation. Isoelectric focusing was done with a Multiphor II (Pharmacia-LKB) by using an Ampholine gel (pH range, 3.5 to 9.5) (Pharmacia-LKB). The group 3 β-lactamase in this study showed a molecular mass of approximately 29 kDa and a pI of approximately 9.5.

The purified enzyme was assayed spectrophotometrically against various β-lactams at 30°C in 50 mM phosphate buffer (pH 7.0) containing 1.0 μM ZnCl₂ (7, 12, 13). K_m and maximum rate of hydrolysis (V_max) were determined with a Lineweaver-Burk plot. V_max/K_m ratios were calculated to compare the effects of β-lactamase on the antibacterial activities of individual β-lactams.

Permeability studies were performed as described in a previous report (16). To raise the production of β-lactamase, an EcoRI-digested fragment of pSW313, including a group 3 β-lactamase gene, was ligated into the EcoRI site of multicopy vector pHSG398. This recombinant, termed pHS313, was introduced into E. coli MC4100. An exponentially growing culture of E. coli MC4100/pHS313 was washed four times in 50 mM phosphate buffer (pH 7.0) containing 1.0 μM ZnCl₂ to remove the enzyme that leaked from damaged cells. The rate of hydrolysis by intact cells in the same buffer with 100 μM substrates was directly measured by a spectrophotometer (12) at 30°C. The supernatant from the intact cell solution was tested to determine the amount of leakage of β-lactamase. The rate of hydrolysis of imipenem by the supernatant was 1.1 to 2.4% of that by the intact cell solution. Hydrolysis of other β-lactams by the supernatant was not detected. The rate of hydrolysis by the intact cell solution was corrected for the contribution made by extracellular enzyme. Permeability coefficients were calculated from the equation of Zimmermann and Rosselet (16).

Kinetic parametric values are shown in Table 1. This enzyme hydrolyzed all β-lactams tested in this study. Ceftazidime and cefpirome showed low relative V_max and high K_m, and piperacillin showed high relative V_max and much higher K_m than other β-lactams; consequently, these agents had low V_max/K_m ratios (range, 0.128 to 0.268). Imipenem showed much higher relative V_max and meropenem had lower K_m than other agents; consequently, these agents had high V_max/K_m ratios (2.67 and 3.22, respectively). As derived from the characterization in this study and a previous report (6), the group 3 β-lactamase from S. marcescens W-313/pSW313 is similar to the IMP-1 metallo β-lactamase that seems to be common in Japan (7, 9, 13).

TABLE 1.

Kinetic parametric values for the group 3 β-lactamase

Substrate	Km (μM)	Relative Vmaxa	Vmax/Km ratio
Ceftazidime	51.7	7.25	0.140
Cefpirome	45.5	12.2	0.268
Meropenemb	4.75	15.3	3.22
Imipenemb	42.7	114	2.67
Piperacillin	439	56.3	0.128

^aRates of hydrolysis are expressed relative to the V_max of cephaloridine, which was set at 100. 

^bKinetic parametric values for meropenem and imipenem cited are from reference 6. 

Table 2 shows the MICs of β-lactams against the transformants of E. coli MC4100, C. freundii GN16922, K. pneumoniae GN19409, and K. oxytoca GN19428, which were transformed with the plasmid encoding the production of the group 3 β-lactamase, and the parent strains. Ceftazidime, cefpirome, and meropenem showed 16- to 500-fold increases in MICs for these transformants in comparison with those for the parent strains, whereas imipenem and piperacillin showed merely 2- to 4-fold increases.

TABLE 2.

Activities against parent strains and transformants carrying the group 3 β-lactamase

Strain	MIC (μg/ml)
	Ceftazidime	Cefpirome	Meropenem	Imipenem	Piperacillin
E. coli MC4100	0.1	0.0125	0.0125	0.1	0.39
E. coli MC4100/pSW313	50	0.78	0.39	0.39	1.56
C. freundii GN16922	0.2	0.025	0.025	0.2	1.56
C. freundii GN16922/pSW313	50	1.56	0.39	0.39	3.13
K. pneumoniae GN19409	0.1	0.025	0.025	0.2	3.13
K. pneumoniae GN19409/pSW313	50	1.56	1.56	0.78	6.25
K. oxytoca GN19428	0.05	0.025	0.025	0.2	1.56
K. oxytoca GN19428/pSW313	25	1.56	0.78	0.78	3.13

Table 3 shows coefficients for permeability to β-lactams of the outer membrane of E. coli MC4100. Ceftazidime and cefpirome exhibited low coefficients for permeability across the outer membrane. As was noted in a previous report by Nikaido et al. (8), the permeability for cefpirome was better than that for ceftazidime. On the other hand, the permeability coefficients for piperacillin and meropenem were eight- to ninefold higher than that for ceftazidime. Moreover, that for imipenem was sixfold higher than those for meropenem and piperacillin. The permeability coefficients for imipenem and meropenem for E. coli MC4100 found in this study were higher than that for S. marcescens S6 reported by Yang et al. (14). S. marcescens S6 may have a much stronger barrier from the outer membrane than E. coli MC4100. Yoshimura and Nikaido found that the permeation rate of piperacillin was <5 by using swelling assay of OmpF porin channel (15), in contrast to our data. Sawai et al. reported that piperacillin exhibited the same MICs against E. coli deficient in OmpF porin and against wild-type E. coli (10). Piperacillin and other β-lactams may permeate into the cell through not only OmpF porin channels but also other channels.

TABLE 3.

Permeability coefficients for β-lactams

Antibiotic	Permeability coefficient (10−5 cm/s)a	Relative permeabilityb
Ceftazidime	0.33 ± 0.11	0.12
Cefpirome	0.70 ± 0.14	0.26
Meropenem	2.97 ± 0.32	1.1
Imipenem	17.49 ± 2.00	6.4
Piperacillin	2.72 ± 0.46	1.0

^aE. coli MC4100/pHS313 was used in permeability studies. 

^bValues are expressed relative to the permeability coefficient for piperacillin, which was set at 1.0. 

Generally, β-lactams show various degrees of activities against gram-negative bacteria with β-lactamases, depending on such properties as the stability to β-lactamases and the permeability of the outer membrane in addition to the affinity to penicillin-binding proteins.

There were distinct differences in the degrees of increase of MICs of β-lactams against transformants with a group 3 β-lactamase-mediating plasmid in comparison with those against the corresponding parent strains. Ceftazidime, cefpirome, and meropenem exhibited 16- to 500-fold decreases in activities against the group 3 β-lactamase producers in comparison with the activities against the parent strains. However, imipenem and piperacillin exhibited merely two- to fourfold decreases in antibacterial activities. As shown by the low V_max/K_m ratios, ceftazidime, cefpirome, and piperacillin were hydrolyzed by this enzyme to a lesser extent than imipenem and meropenem. The stability to the group 3 β-lactamase did not directly accord with the increases of MICs of the β-lactams. This contradiction could be explained on the basis of the permeability study of E. coli MC4100. Ceftazidime and cefpirome elicited low permeability of the outer membrane, but permeability coefficients for piperacillin and meropenem were eight- to ninefold higher than that for ceftazidime. Moreover, permeability to imipenem was sixfold higher than those to meropenem and piperacillin. The low permeabilities enhanced the effects of β-lactamase on the antibacterial activities, as shown for ceftazidime and cefpirome, while the extremely high permeability to imipenem reduced the effect of the β-lactamase. The permeability to meropenem could not sufficiently compensate for the reduction in the amount of agent caused by the efficient hydrolysis by β-lactamase. On the other hand, the high permeability to piperacillin, in addition to the reduced amount of hydrolysis, supplies high concentrations of the agent to inhibit the penicillin-binding proteins in the periplasmic space.

Cornaglia et al. reported (2), in contrast to our data, that the MICs of meropenem and ceftazidime against E. coli LGC10, which produces a carbapenem-hydrolyzing β-lactamase (CphA), were equal to those against the parent strain, which lacks CphA, and E. coli LGC10 exhibited much higher permeability coefficients than those found in our study. On the basis of our theory, the very weak barrier of the outer membrane in E. coli LGC10 may permit meropenem and ceftazidime to exhibit low MICs against a CphA producer, equivalent to the MICs against CphA nonproducers.

For C. freundii GN16922/pSW313, K. pneumoniae GN19409/pSW313, and K. oxytoca GN19428/pSW313 as well, there were distinct differences in the levels of increase of the MICs. These phenomena among β-lactams could be attributed to the role of permeability, as was demonstrated with E. coli MC4100/pSW313.

In the future, increasing numbers of clinical strains may acquire the characteristic of production of a group 3 β-lactamase. If the outer membranes of gram-negative bacteria with this enzyme provide the typical barrier, these bacteria would be susceptible to β-lactams with superior permeation and moderate resistance to hydrolysis by this enzyme.