Hans Herbert Martin

The signal transducer encoded by ampG is essential for induction of chromosomal AmpC β-lactamase in Escherichia coli by β-lactam antibiotics and ‘unspecific’ inducers

Chemical mutagenesis of the AmpC beta-lactamase-hyperinducible Escherichia coli strain SN0301/pNu305 carrying the cloned ampC and ampR genes from Citrobacter freundii OS60 gave four independent mutants in which beta-lactamase was no longer inducible, or was inducible only to a low level, by beta-lactam antibiotics. The genes ampC, ampR, ampD and ampE, which were essential for beta-lactamase induction, were functional in these mutants. In all four mutants, the sites of mutation were mapped to 9.9 min on the E. coli chromosome. Complementation with wild-type ampG restored inducibility of beta-lactamase to wild-type levels. The nucleotide sequence of all four mutant ampG alleles (ampG1, ampG3, ampG4 and ampG5) was determined. In three of the mutants, a single base exchange led to an amino acid change from glycine to aspartate at different sites in the deduced amino acid sequence. In the fourth mutant (ampG4), with low-level inducibility, the nucleotide sequence was identical to wild-type ampG. Spontaneous back-mutation of the chromosomal ampG1 mutant resulted in restoration of wild-type inducibility and a return to the wild-type ampG sequence. Unspecific induction by components of the growth medium was also dependent on intact ampG function.

State of penicillin-binding proteins and requirements for their bactericidal interaction with β-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum β-lactams

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum beta-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum beta-lactam antibiotics and with constitutively overproduced chromosomal beta-lactamase in the periplasm. The binding of structurally different beta-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1.5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested beta-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced beta-lactamase did not prevent PBP-beta-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific beta-lactams.

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.