W E Sanders

Selection of multiple antibiotic resistance by quinolones, beta-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes.

The ability of three quinolones, two beta-lactams, and one aminoglycoside to select resistant mutants was examined in tests with 30 isolates of commonly encountered nosocomial pathogens. Ciprofloxacin and norfloxacin, two new quinolone derivatives, were no more likely to select resistant mutants than amikacin, whereas nalidixic acid, an older quinolone derivative, was the most likely of the six drugs examined to select resistant mutants. Mutational frequencies of 10(-7) to 10(-8) were observed in most instances. In general, the mutants were 8 to 16 times less susceptible to the drug used for selection. Although most quinolone-selected mutants were cross-resistant only to other drugs within this class, certain mutants of Klebsiella pneumoniae selected by nalidixic acid, ciprofloxacin, or norfloxacin were also less susceptible to beta-lactam antibiotics. This unusual pattern of multiple drug resistance was associated with changes in outer membrane proteins of the organism. Multiple drug resistance was also observed in beta-lactam-selected mutants of Enterobacter cloacae and Pseudomonas aeruginosa (beta-lactams), amikacin-selected mutants of Providencia stuartii and P. aeruginosa (aminoglycosides), and beta-lactam- or amikacin-selected mutants of Serratia marcescens (beta-lactams plus aminoglycosides). These results underscore the need to examine carefully the frequency with which resistance to any new antibiotic develops, as well as the patterns of multiple drug resistance which may occur simultaneously. Images

In vitro antagonism of beta-lactam antibiotics by cefoxitin.

We assessed the extent and mechanisms of antagonism of beta-lactam antibiotics by cefoxitin. In tests with 41 gram-negative isolates, cefoxitin antagonized cephalothin, cefamandole, cefsulodin, cefotaxime, moxalactam, ampicillin, carbenicillin, piperacillin, mezlocillin, and azlocillin, but not cephalexin, mecillinam, or N-formimidoyl thienamycin. The extent of antagonism varied with the beta-lactam and genus studied. However, antagonism occurred most often with strains possessing inducible cephalosporinases. Antagonism of cephalothin and cefamandole correlated closely with the induction of beta-lactamases capable of inactivating these drugs. Although antagonism of the remaining drugs occurred more often with strains possessing inducible beta-lactamases, these enzymes did not inactivate the drugs. Morphological studies revealed that cefoxitin inhibited filamentation and lysis produced by various beta-lactam drugs. Results of this investigation suggest that cefoxitin antagonizes beta-lactams via (i) induction of drug-inactivating beta-lactamases, and (ii) the induction of beta-lactamases that cannot inactivate the drug but serve as barriers against access to target proteins. This barrier appears most efficient for drugs that bind to penicillin-binding proteins 1 and 3. Images

Beta-lactamase production in members of the family Enterobacteriaceae and resistance to beta-lactam-enzyme inhibitor combinations.

Recent reports that members of the family Enterobacteriaceae that produce high levels of certain beta-lactamases are often resistant to ticarcillin-clavulanate prompted this study to assess the relationship between type and amount of enzyme produced and susceptibility to ticarcillin-clavulanate, piperacillin-tazobactam, and cefoperazone-sulbactam. Agar dilution MICs were determined by using 73 strains of Enterobacteriaceae that produced a single beta-lactamase that had been characterized and quantified and a beta-lactamase-negative control strain of Escherichia coli. For E. coli and Klebsiella pneumoniae, MICs of each combination increased as levels of TEM, SHV-1, or class IV enzymes increased. However, the percentage of strains that were resistant was highest for ticarcillin-clavulanate (32%), with only 18 and 6% resistant to piperacillin-tazobactam and cefoperazone-sulbactam, respectively. Strains producing PSE-1, regardless of level, were resistant or moderately susceptible to ticarcillin-clavulanate but were susceptible to piperacillin-tazobactam and cefoperazone-sulbactam. HMS-1 and OHIO-1 beta-lactamases were associated with resistance to ticarcillin-clavulanate and piperacillin-tazobactam, respectively. High levels of class IV enzymes in Klebsiella oxytoca were associated with resistance to all three combinations. These results indicate that the level and type of beta-lactamase produced by members of the family Enterobacteriaceae are important determinants of susceptibility to beta-lactam-inhibitor combinations, especially ticarcillin-clavulanate.

Susceptibility of Organisms in the Mycobacterium fortuitum Complex to Antituberculous and Other Antimicrobial Agents

Of 21 antimicrobial agents tested in vitro, amikacin was the most predictably active against clinical isolates belonging to the Mycobacterium fortuitum complex; however, only 50% of strains studied were susceptible to clinically attainable concentrations of the drug.

Antagonism of carbenicillin and cefamandole by cefoxitin in treatment of experimental infections in mice.

The ability of cefoxitin to antagonize the in vivo efficacy of cefamandole and carbenicillin as predicted by in vitro assays was analyzed in experimental infections in mice. Cefoxitin was administered in a nonprotective dose either at the time of challenge or simultaneously with the protective drug, 1 and 3.5 h postchallenge. In mice infected with Enterobacter cloacae, median 50% protective doses of cefamandole and carbenicillin were markedly increased by cefoxitin, especially when the latter was given at the time of challenge. The antagonistic effect was also associated with increased numbers of challenge bacteria present in animal heart blood within a 6.5-h period after infection. In infections with Pseudomonas aeruginosa, cefoxitin antagonized carbenicillin; however, the effect was less dramatic than that seen with E. cloacae. Antagonism in this model was pronounced with simultaneous administration of antagonizing and protective drugs. The antagonistic effects observed in all in vivo tests were not due to the selection of stable resistance to the protective drugs, but appeared to be due to a reversible induction of beta-lactamases by cefoxitin.

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

The chromosomal beta-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 beta-lactamase expression, whether derived in the laboratory or recovered from patients treated with one of the new beta-lactam antibiotics, were found to have no detectable alterations in outer membrane proteins. Derepression of beta-lactamase in these mutants was associated with high-level resistance to multiple beta-lactam antibiotics, while loss of inducible beta-lactamase (i.e., production of basal enzyme levels only) was associated with acquisition of susceptibility to many beta-lactam antibiotics, including cephalothin. In contrast, alteration in outer membrane proteins was associated with only moderate-level resistance to beta-lactam antibiotics. However, this included resistance to such drugs as amdinocillin and Sch 34343, which were unaffected by derepression of beta-lactamase. 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 beta-lactamase and outer membrane proteins can affect the susceptibility of E. cloacae to many antibiotics. However, alterations in beta-lactamase alone are sufficient to produce high-level multiple beta-lactam resistance in this organism. Images

Evaluation of single-dose ciprofloxacin in the eradication of Neisseria meningitidis from nasopharyngeal carriers.

The ability of a single oral 750-mg dose of ciprofloxacin to eradicate Neisseria meningitidis from persistent nasopharyngeal carriers was prospectively evaluated in a placebo-controlled, randomized, double-blinded study. Cultures of specimens taken from all 23 ciprofloxacin-dosed subjects 1 day postdose were negative; cultures from 96% of these subjects were negative at 7 and 21 days postdose, including a specimen from a subject colonized with a minocycline-resistant strain. Of 22 placebo recipients, 20 (91%) remained culture positive. Single-dose ciprofloxacin appears efficacious for meningococcal prophylaxis.

Evidence for multiple forms of type I chromosomal beta-lactamase in Pseudomonas aeruginosa.

The multiple stages of derepression of the type I chromosomal beta-lactamase in Pseudomonas aeruginosa were examined. Mutants partially and fully derepressed for beta-lactamase were selected from a wild-type clinical isolate. An analysis of the beta-lactamase produced by these mutants and the induced wild type revealed significant differences in the products of derepression at each stage. Beta-lactamase produced by the fully derepressed mutant showed a lower affinity (Km, 0.113 mM) for cephalothin than that produced by the partially derepressed mutant (Km, 0.049 mM). However, due to a very large Vmax, the former possessed a much greater hydrolytic efficiency. Differences in substrate profile were also noted. Only beta-lactamase from the fully derepressed mutant hydrolyzed cefamandole, cefoperazone, and cefonicid. The partially derepressed mutant possessed a single beta-lactamase band with a pI of 8.4. The fully derepressed mutant possessed this band and an additional major band with a pI of 7.5. Induction of the wild type with cefoxitin produced both bands. The changes in physiologic parameters of the enzymes produced in the different stages of derepression suggest a complex system for beta-lactamase expression in P. aeruginosa. This may involve at least two distinct structural regions, each of which is under control of the same repressor. Images

In Vitro Studies with Sch 21420 and Sch 22591: Activity in Comparison with Six Other Aminoglycosides and Synergy with Penicillin Against Enterococci

In vitro tests were performed with Sch 21420 and Sch 22591 to determine (i) their activity in comparison to six other aminoglycosides against 343 clinical isolates, and (ii) whether synergy with penicillin G could be demonstrated with enterococci. In broth dilution tests, Sch 22591 was more active than the seven other aminoglycosides against Staphylococcus aureus, Enterobacteriaceae, and most nonfermenting gram-negative bacilli. Sch 22591 was as active as tobramycin against Pseudomonas aeruginosa. The activity of Sch 21420 was comparable to gentamicin, sisomicin, netilmicin, and tobramycin but greater than amikacin or kanamycin against S. aureus and most genera of Enterobacteriaceae. Sch 21420, amikacin, and kanamycin were (i) more active than the other five aminoglycosides against Proteus rettgeri and Providencia stuartii, but (ii) less active than the other five aminoglycosides against Neisseria gonorrhoeae, enterococci, most nonfermenting gram-negative bacilli, Proteus mirabilis, and Proteus morganii. Studies on the bactericidal activity of Sch 22591 with penicillin indicated a synergistic interaction against enterococci, including strains highly resistant to streptomycin and kanamycin. This could be demonstrated with combinations containing 3.0 to 6.0 μg of Sch 22591 per ml and was comparable to that observed with penicillin/gentamicin. Penicillin plus Sch 21420 (25 μg/ml) also demonstrated synergy against enterococci, including strains highly resistant to streptomycin. However, synergy did not occur against strains highly resistant to kanamycin. These latter results were similar to those obtained in tests with penicillin/kanamycin.

Heterogeneity of class I beta-lactamase expression in clinical isolates of Pseudomonas aeruginosa.

Expression of chromosomal beta-lactamase was examined in 85 clinical isolates of Pseudomonas aeruginosa. beta-Lactamase assays with and without cefoxitin induction revealed four phenotypes of enzyme expression: low basal, inducible; moderate basal, inducible; moderate basal, constitutive; and high basal, constitutive. The isoelectric points of the major beta-lactamase bands were 9.4, 9.2, and 8.4. These results indicate that there is a limited heterogeneity in expression of chromosomal beta-lactamase of P. aeruginosa. Images