Involvement of Fe Uptake Systems and AmpC β-Lactamase in Susceptibility to the Siderophore Monosulfactam BAL30072 in Pseudomonas aeruginosa

Abstract

BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC ␤-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16؋ the MIC in iron-sufficient conditions. At 4؋ to 8؋ the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8-to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 g/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8-and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072.

Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy

“…The fecI gene is an extracytoplasmic function (ECF) sigma-70 factor that acts as a transcriptional activator of the ferric citrate siderophore system (22). Mutations at this pfecI location have been reported for BAL30072-resistant isolates, and it was shown that by derepressing the fecIRA operon, a mutation in the Fur-binding promoter of fecI led to increased expression of the ferric citrate siderophore receptor FecA (20). Transcriptional analysis of our C2-ARC3502 pfecI mutant yielded the same result of elevated fecA expression relative to the parent (data not shown).…”

“…For five mutants, a variety of genetic changes were seen in the piuA and piuC genes, including insertions, deletions, and frameshifts all likely to result in a loss of function of the encoded proteins. PiuA is an outer membrane siderophore receptor with similarity to the Fiu catechol receptor, while PiuC is a predicted Fe(II)-dependent oxygenase with its gene located directly adjacent to PiuA (19,20). The predicted effect of a piuA or piuC mutation is decreased expression of the PiuA receptor, which is one of two receptors that hydroxypyridinone-based siderophore conjugates are believed to exploit for entry, with the other being PirA (20,21).…”

“…PiuA is an outer membrane siderophore receptor with similarity to the Fiu catechol receptor, while PiuC is a predicted Fe(II)-dependent oxygenase with its gene located directly adjacent to PiuA (19,20). The predicted effect of a piuA or piuC mutation is decreased expression of the PiuA receptor, which is one of two receptors that hydroxypyridinone-based siderophore conjugates are believed to exploit for entry, with the other being PirA (20,21). A frameshift mutation was also found in the gene encoding PirR, which is a new resistance mechanism to be reported for a siderophore-conjugated ␤-lactam.…”

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

“…The close phylogenetic relationship between Acinetobacter and Pseudomonas prompted us to search in A. baumannii for orthologues of the previously identified TonB-dependent receptors PiuA and PirA from P. aeruginosa (15,31). For each of these proteins, we identified one orthologue (Ͼ40% amino acid identity).…”

Urinary Concentrations and Antibacterial Activity of BAL30072, a Novel Siderophore Monosulfactam, against Uropathogens after Intravenous Administration in Healthy Subjects

“…This monocyclic ␤-lactam contains an iron-chelating dihydroxypyridone substituent. Using a transcriptome approach, the FecIRA Fe 3ϩ -dicitrate transporter was identified as a regulatory cascade, and the PiuA iron receptor was identified as a putative uptake system affecting susceptibility to BAL30072 (3). The molecule has potent in vitro activity against many species of MDR isolates of the Enterobacteriaceae family, including isolates harboring a class A carbapenemase or a metallo-␤-lactamase (4,5).…”