Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

Abstract

b Amdinocillin (mecillinam) is a ␤-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 ؋ 10 ؊8 to 2 ؋ 10 ؊5 per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli. Because of the rapid increase in antibiotic resistance, several first-line antibiotics have been rendered less suitable for empirical treatment. For example, in Sweden, the increasing resistance to trimethoprim has led to a shift in the empirical therapy of uncomplicated urinary tract infections (UTIs), with nitrofurantoin and pivmecillinam now being suggested as first-line therapy (1). It is also recommended as a first-line UTI treatment by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases (2). Amdinocillin (Amd; also known as mecillinam) is an extended-spectrum penicillin derivative, a 6␤-amidinopenicillanic acid developed in the 1970s (3, 4). It has been widely used in the Scandinavian countries for UTI treatment since the early 1980s (5, 6). A large proportion of the drug is excreted unchanged in the urine (1, 7), leading to high concentrations in urine and a limited effect on the commensal flora (1,8,9). Amd binds to and inhibits the transpeptidase activity of penicillin-binding protein 2 (PBP2) (10-13). PBP2 is responsible for the elongation of rod-shaped cells, and cells treated with Amd become enlarged, nondividing spheres that ultimately lyse (11,14).Resistance development has remained low in countries where it is used (15-17). This finding is seemingly a paradox, ...

Interactions of OP0595, a Novel Triple-Action Diazabicyclooctane, with β-Lactams against OP0595-Resistant Enterobacteriaceae Mutants

“…Aside from OP0595 resistance, their most consistent trait was sharply increased resistance to amdinocillin, which solely targets PBP2, and smaller MIC increases for imipenem, which primarily targets PBP2 (18,19). Recent studies show that amdinocillin selects diverse mutations, mostly upregulating the stringent response and increasing cellular levels of guanosine tetraphosphate (20), and preliminary data suggest similar patterns among the present OP0595-selected mutants (21). Changes to pbp2 itself are rare or absent.…”

“…Changes to pbp2 itself are rare or absent. It is unclear how ppGpp-induced changes protect against PBP2-active agents (20,22), but it is most plausible that they are somehow compensatory. Notably, amdinocillin-and OP0595-resistant mutants grow as stable round forms under amdinocillin or OP0595 challenge (1,6,23), indicating that PBP2 itself remains vulnerable, an observation keeping with the retention of the enhancer effect.…”

In Vitro and In Vivo Activities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae

“…These findings suggested that some strains of Enterobacteriaceae were resistant to the antibacterial activity of OP0595 due to PBP2 inhibition and that these OP0595-resistant strains of Enterobacteriaceae subsequently became the major population. This resistance to PBP2 inhibitors occurs readily, but the fitness cost for the resistant clones that survive is high, as shown by a study by Doumith et al and a study of mutants with resistance to amdinocillin, another PBP2 inhibitor (19,20). In addition, OP0595-resistant Enterobacteriaceae spp.…”

Tackling antimicrobial resistance in lower urinary tract infections: treatment options

“…The mechanism(s) of resistance to mecillinam are currently poorly understood although a recent study showed that mutation in cysB preventing the production cysteine was found in clinical isolates. The reduced cysteine levels led to an increased cellular concentration of the ppGpp molecule, which makes the drug's PBP2 target nonessential [120].…”