Role of beta-lactamase in in vivo development of ceftazidime resistance in experimental Pseudomonas aeruginosa endocarditis

Abstract

Two ceftazidime-resistant variants of Pseudomonas aeruginosa (PA-48, PA-60), obtained from cardiac vegetations of rabbits with endocarditis receiving ceftazidime therapy, were studied for mechanisms of resistance. Both resistant variants were stably derepressed for the type Id I-lactamase, which was ceftazidime inducible in the parental strain (PA-96) used to initially infect the rabbits. There was no evidence of ceftazidime bioinactivation by the resistant strains, and their outer membrane permeabilities were comparable to those of the parental strain. No alterations were observed in patterns of outer membrane proteins or membrane lipopolysaccharides in the resistant variants as compared with the parental strain. Penicillin-binding protein patterns of the resistant variants revealed the absence of penicillin-binding protein 4 in both, with acquisition of a new protein of higher apparent molecular weight in PA-60. Calculation of the rate of appearance of ceftazidime in the periplasm at sub-MICs suggested that slow enzymatic hydrolysis of the P-lactam, rather than nonhydrolytic trapping, was the major explanation for the induced resistance in vivo in strains PA-48 and PA-60.,-Lactam antibiotics are of primary use in the therapy of gram-negative bacterial infections. However, the appearance of bacterial resistance to successive generations of P-lactams (30,36) has necessitated the continuous development and assessment of new compounds. The major impediments to ,-lactam therapy have been the high intrinsic resistance of some organisms, including Pseudomonas aeruginosa due to the intrinsic barrier properties of their outer membranes (21, 39) and the acquisition of high levels of P-lactamase due either to the presence of a plasmid or to the derepression of a formerly inducible, chromosomally encoded , B-lactamase (30, 34, 36). Mutational alterations in the 3-lactam targets, penicillin-binding proteins (PBPs), have also been observed (26) but seem to occur with somewhat less frequency. One novel approach taken by pharmaceutical companies in the development of newer ,-lactam antibiotics has been to search for compounds that are refractory to ,-lactamase-mediated hydrolysis. Recently, however, it has been observed that, despite the extremely low rates at which these compounds are hydrolyzed, clinical isolates of P. aeruginosa and Enterobacter cloacae that are resistant to these so-called P-lactamase-stable P-lactams often have much-increased levels of the chromosomal P-lactamase (4,30,31,(34)(35)(36). The mechanism of resistance has been suggested to involve nonhydrolytic binding of the ,-lactam by molecules of ,-lactamase, thus reducing the periplasmic concentration (i.e., the concentration in the vicinity of the target PBPs) to subinhibitory levels (34). Alternatively, Vu and Nikaido (35) have argued convincingly that a combination of low-level hydrolysis by ,-lactamase (low Km. low Vmax) and reduced outer membrane permeability, thus di-* Corresponding author. t Present address:

Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between β‐lactamase‐mediated trapping and hydrolysis as mechanisms of resistance

“…The data in Table 3 also agree with the outer-membrane-permeability parameters of 0.0015 to 0.0035 cm3 min-' mg dry mass-' measured for nitrocefin in P . aeruginosa [23]. The permeability parameters for cefsulodin given in Table 3 refute the hypothesis 1281 that cefsulodin diffuses across the outer membrane especially rapidly in P. aeruginosa.…”

“…That is, each generation of cells would have to synthesize an extra P-lactamase complement of 41% of its own mass. We suggest that this is unlikely, as did Bayer et al [23] on the basis of a similar calculation. The following argument strengthens the case against the trapping hypothesis.…”

“…Nevertheless, in the absence of such a direct measurement, the measurements reported here (Table 3) and elsewhere [23, 561 should be adequate as a first approximation. This was the analytical approach taken by Vu and Nikaido [17] in their study of trapping and hydrolysis in Enterobacter cloacae, by Bayer et al [23] in their similar work on P . aeruginosa and ceftazidime, and by Nikaido and Normark [21] in their experimental test of the kinetic theory [19,20,22] of the fl-lactam MIC.…”

“…Even in that study, permeabilities of the outer membranes of whole cells for second-and third-generation cephalosporins were not measured directly, but estimated from the more-easily-measured permeability to cefazolin [17], a first-generation compound [l]. The second study was of ceftazidime resistance associated with elevated p-lactamase synthesis in Pseudomonas aeruginosa [23]. Again, however, these authors did not measure outer-membrane permeabilities to ceftazidime directly but assumed that they would be identical to permeabilities to nitrocefin.…”

Bactericidal interactions of a beta-lactam and beta-lactamase inhibitors in experimental Pseudomonas aeruginosa endocarditis caused by a constitutive overproducer of type Id beta-lactamase

“…Such resistances have been variably directed towards the aminoglycoside or P-lactam components of the combination therapy regimens generally used to treat Pseudomonas endocarditis (1,19,23,26). The mechanism of P-lactam resistance in these situations has usually been associated with either inducible or constitutive P-lactamase overproduction (3,19,26).One possible strategy to circumvent the above P-lactamase-related resistances is to interfere with the function of the enzyme by using P-lactamase inhibitors. The present study was designed to (i) evaluate several P-lactamase inhibitors in vitro for their ability, when combined with a P-lactam agent (ceftazidime), to synergistically kill a ceftazidime-resistant strain of P. aeruginosa which constitutively overproduces ,-lactamase; (ii) define the ability of P-lactamase inhibitors to specifically abrogate enzyme activity in vitro; and (iii) determine the in vivo effect of combinations of P-lactam plus P-lactamase inhibitors in experimental endocarditis caused by the variant, P. aeruginosa PA-48, which conistitutively overproduces type Id 1B-lactamase.…”