Bacillus megaterium resistance to cloxacillin accompanied by a compensatory change in penicillin binding proteins

Abstract

BACTERIA have been shown to become resistant to β-Lactam antibiotics (penicillin and cephalosporins) by the following methods: decreased permeability^1,2, increased production of an enzyme (β-Lactamase) which degrades the antibiotics¹, acquisition of a plasmid that produces a β-Lactam^3,4, and a decreased affinity of the lethal target for the antibiotic⁵. β-Lactam antibiotics are thought to kill bacteria by interfering with the terminal stages of cell wall biosynthesis⁶. The cross-linking of the cell wall by a transpeptidase enzyme has been implicated as the probable target site^7,8, although inhibition of a carboxypeptidase⁹ and stimulation of autolytic activity¹⁰ have also been proposed. It has been suggested that penicillin is a transition state analogue of the D-alanyl-D-alanine moiety of the donor molecule in the transpeptidation reaction¹¹and that penicillin binds covalently to the transpeptidase⁷. Investigations therefore concentrated on enzymes which could fix penicillin in bacterial membranes^12,13. Bacteria contain multiple penicillin binding proteins (PBPs) and it is important to identify which one is the lethal target. One approach used in this laboratory has been to purify the individual PBPs and investigate their enzymatic activity, although it was realised that solubilisation might lead to inactivation. In Bacillus megaterium the only PBP shown to possess enzymatic activity was PBP 5 (ref. 14) (the one of lowest molecular weight), and this was the D, D-Ala carboxypeptidase, which is much less sensitive to β-lactams than is growth of the organism¹⁵. Despite the lack of enzymatic activity the affinity of the remaining PBPs 1–4 for benzylpenicillin could be measured in whole cells. PBP 1 was the only protein that interacted covalently with benzyl-penicillin at the lowest concentration of the antibiotic that would inhibit growth, and it was therefore proposed to be the lethal target¹⁶. An alternative approach is to determine which PBPs become altered as an organism develops resistance to an antibiotic. This method has shown that resistance can be due to a PBP having a decreased affinity for a β-Lactam antibiotic. Using this technique PBP 2 has been identified as the lethal target for mecillinam in Escherichia coli⁵ and for cloxacillin in Bacillus subtilis¹⁷. However, in this report a new mode of resistance is described in which there is a purely quantitative change in the ratio of PBP 1 to PBP 3 as resistance to cloxacillin develops in B. megaterium. Thus, resistance results from a decrease in the level of the lethal target which is compensated by an increase in a less sensitive target.