Mutational Enzymatic Resistance of Enterobacter Species to Beta-Lactam Antibiotics

The chromosomal I-lactamase and outer membrane proteins of Enterobacter cloacae were examined to determine their relative contributions to multiple antibiotic resistance in this organism. Mutants altered in I(-lactamase expression, whether derived in the laboratory or recovered from patients treated with one of the new ,-lactam antibiotics, were found to have no detectable alterations in outer membrane proteins. Derepression of ,I-lactamase in these mutants was associated with high-level resistance to multiple I-lactam antibiotics, while loss of inducible ,-lactamase (i.e., production of basal enzyme levels only) was associated with acquisition of susceptibility to many ,-lactam antibiotics, including cephalothin. In contrast, alteration in outer membrane proteins was associated with only moderate-leVel resistance to P-lactam antibiotics. However, this included resistance to such drugs as amdinocillin and Sch 34343, which were unaffected by derepression of P-lactalnase. Resistance to chloramphenicol and tetracycline also accompanied changes in outer membrane proteins. Although the outer membrane proteins of various strains of E. cloacae were similar, there did appear to be some major strain-to-strain variations. Thus, it appears that alterations in both 13-lactamase and outer membrane proteins can affect the susceptibility of E. cloacae to many antibiotics. However, alterations in P-lactamase alone are sufficient to produce high-level multiple 3-lactam resistance in this organism.Mutants of Enterobacter spp. producing high levels of P-lactamase have been shown to be responsible for a number of clinical failures associated with emergence of resistance during therapy with the newer expanded-spectrum ,-lactam antibiotics (16). These mutants occur spontaneously at a frequency of 10-6 to 10-7 and appear to have lost control of P-lactamase production (8, 10). Normally, production of the chromosomally mediated cephalosporinase of Enterobacter spp. is under repressor control (8). Loss of this normal control mechanism via mutation leads to high-level Ilactamase production. This event is associated with resistance to multiple P-lactam antibiotics (8, 10, 14, 16). This resistance has been difficult to explain solely on the basis of P-lactamase-mediated hydrolysis because it involves many drugs that appear to be poor substrates for the enzyme. Thus, it has been speculated that this resistance must also involve a change in permeability.The permeability of gram-negative organisms to ,B-lactam antibiotics is determined primarily by outer membrane proteins (13). Studies by Sawai et al. (17) and Kaneko et al. (9) indicate that in Enterobacter cloacae two major outer membrane proteins appear to be involved in cephalosporin permeation. Alterations in these proteins have been shown to be associated with multiple P-lactam resistance in this organism (17). Since changes in P-lactamase expression and outer membrane proteins can be responsible for multiple 13-lactam resistance, this investigation was designed to examine both these fact...

mentioning

confidence: 99%

“…These mutants occur spontaneously at a frequency of 10-6 to 10-7 and appear to have lost control of P-lactamase production (8,10). Normally, production of the chromosomally mediated cephalosporinase of Enterobacter spp.…”

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confidence: 99%

Role of beta-lactamases and outer membrane proteins in multiple beta-lactam resistance of Enterobacter cloacae

Werner¹,

Sanders²,

Sanders³

et al. 1985

“…Strains reversibly derepressed by cefoxitin or stably derepressed via mutation become resistant not only to the newer cephalosporins but also to expanded-spectrum penicillins (5, 10,17,27,29,30,32; C. C. Sanders and W. E. Sanders, Jr., Rev. Infect.…”

mentioning

confidence: 99%

Characterization of beta-lactamase induction in Enterobacter cloacae

Gootz¹,

Sanders²

1983

The induction of ,-lactamase was studied in a strain of Enterobacter cloacae. A wide variety of P-lactam compounds were found to induce ,-lactamase in this organism, and the degree of induction was directly related to the stability of the inducer to degradation by the enzyme. The kinetics of the induction process were consistent with a system normally under repressor control, suggesting a direct interaction of the P-lactam compound with a repressor protein in the E. cloacae cells. Although these characteristics are common to many inducible systems in gram-negative organisms, the induction of 1-lactamase in this strain was not subject to catabolite repression with glucose and remained unaffected by exogenous cyclic AMP in the culture medium. This suggests that the organization and function of the P-lactamase regulatory genes in E. cloacae are unlike those of other inducible gene systems, such as those composing the well-characterized lactose operon in Escherichia coli.

“…Resistance is reported to, develop to most recent P-lactams, with the exception of those such as mecillinam and imijOikm (N-forminiidoyl thienamycin) that bind primarily to penicillin-binding protein 2 (10). The rapidity and extent of such resistance is of major concern and could lead to curtailment of the tise of second-and thirdgeneration cephalosporins and antipseudomonal penicillins.The basis of such resistance has been proposed to be due to a derepression or mutation to stable derepressed (cOnstitutive) production of the inducible -lactamase (3,4,6,10). Resistance is achieved by either hydrolysis of p-lactams or by a nonhydrolytic barrier for those not subject to hydrolysis (10, 13).…”

mentioning

confidence: 99%

“…The basis of such resistance has been proposed to be due to a derepression or mutation to stable derepressed (cOnstitutive) production of the inducible -lactamase (3,4,6,10). Resistance is achieved by either hydrolysis of p-lactams or by a nonhydrolytic barrier for those not subject to hydrolysis (10, 13).…”

mentioning

confidence: 99%

Resistance of Pseudomonas aeruginosa mutants with altered control of chromosomal beta-lactamase to piperacillin, ceftazidime, and cefsulodin

Bryan¹,

Kwan²,

Godfrey³

1984

Examination of 3-lactam susceptibility of mutants altered in the control of chromosomal J3-lactaimase of Pseudomonas aeruginosa supports the view that a constitutive level of P-lactamiase is not an adequate explanation for resistance to cefsulodin and ceftazidime but could be for resistance to piperacillin which has an efficiency of hydrolysis ca. 10 times higher than does cefsulodin or ceftazidime.In initial studies of expanded&spectrum cephalosporins and penicillins, the rapid development of resistance to many of these agents has beeh reported among Pseudomonas, Enterobacter, and Serratia strains which possess inducible ,3-lactainases (10). Resistance is reported to, develop to most recent P-lactams, with the exception of those such as mecillinam and imijOikm (N-forminiidoyl thienamycin) that bind primarily to penicillin-binding protein 2 (10). The rapidity and extent of such resistance is of major concern and could lead to curtailment of the tise of second-and thirdgeneration cephalosporins and antipseudomonal penicillins.The basis of such resistance has been proposed to be due to a derepression or mutation to stable derepressed (cOnstitutive) production of the inducible -lactamase (3,4,6,10). Resistance is achieved by either hydrolysis of p-lactams or by a nonhydrolytic barrier for those not subject to hydrolysis (10, 13). In the latter case it has been proposed that 1-lactamase binds P-lactam molecules and prevents them from bindihg to target penicillin-binding proteins. Although it is clear that some of the resistant isolates are constitutive mutants, it has not yet been cohfirmed that constitutive production is the only mechanism operating to produce resistance, particularly to nonhydrolyzed -lactams in the various strains isolated to date. Recently, Seeberg et al. (11) showed by genetic transfer experiments to Escherichia coli that chromosomal 13-lactamases of Enterobacter cloacae are responsible for resistance to expanded-spectium cephalosporins. However, details of the transfer of the resistance gene(s) were not provided so it is not yet possible to conclude that only a single gene product is involved. To determine whether constitutive P-lactaniase is the sole explanatidh for this type of resistance in! Pseudomonas aeruginosa, we examined a set of isogenic mutants of a bacterium obtained from H. Matsumoto (7); these mutants vary in the control of chromosomal P-lactamase. The P.. aeruginosa strains used were PA04069 met-9020 pro-9024 (normally inducible), PA04082 met-9020 pro-9024 blaJ91ll (cohstitutive), and PA04098 met-9020 pro-9024 blaP-9208 (weaklactamfnase producer).We confirmed that this set of P. aeruginosa strains and strain PA01 have identical patterns of outer membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2), penicillin binding (2), and lipopolysaccharide binding (5)