Clinically Relevant Gram-Negative Resistance Mechanisms Have No Effect on the Efficacy of MC-1, a Novel Siderophore-Conjugated Monocarbam

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.

Section: Discussionmentioning

confidence: 89%

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

Delden

Page

Köhler

2013

“…This catechol moiety contributes to potent antimicrobial activity against Gram-negative bacteria by functioning as a siderophore to form a chelating complex with ferric iron (14). As reported for other ␤-lactam antibiotics with iron chelating siderophore moiety, the enhanced antimicrobial activity is related to the active outer membrane transport into the periplasmic space where the ␤-lactam antibiotic efficiently inhibits cell wall synthesis (15)(16)(17)(18). In addition, S-649266 is more stable against various ␤-lactamases, including KPC-3 and NDM-1 carbapenemases, than ␤-lactam agents such as cefepime (FEP) and meropenem (MEM) (19).…”

mentioning

confidence: 92%

“…The apo-transferrin was added to mimic the ferric-iron-limiting condition in human biological fluids in which free iron is tightly bound to proteins such as transferrin (21). Determination of MIC values in iron-deficient conditions has been used to evaluate the activities of other siderophore antibiotics (14,16,17,22). S-649266 was synthesized at Shionogi & Co., Ltd. (Osaka, Japan).…”

Section: Bacterial Strainsmentioning

confidence: 99%

In Vitro Antimicrobial Activity of a Siderophore Cephalosporin, S-649266, against Enterobacteriaceae Clinical Isolates, Including Carbapenem-Resistant Strains

Kohira

West

Ito

et al. 2016

213

181

Enterobacter cloacae isolates were all <1 g/ml, and there were only 8 isolates (1.3%) among these 617 clinical isolates with MIC values of >8 g/ml. In the second set, the MIC values of S-649266 were <4 g/ml against 109 strains among 116 KPC-producing and class B (metallo) carbapenemase-producing strains. In addition, S-649266 showed MIC values of <2 g/ml against each of the 13 strains that produced other types of carbapenemases such as SME, NMC, and OXA-48. The mechanisms of the decreased susceptibility of 7 class B carbapenemase-producing strains with MIC values of >16 g/ml are uncertain. This is the first report to demonstrate that S-649266, a novel siderophore cephalosporin, has significant antimicrobial activity against Enterobacteriaceae, including strains that produce carbapenemases such as KPC and NDM-1.

“…This mutant was constructed by replacing the C terminus of PvdR (amino acids 68 to 691), all of PvdT, and the majority of OpmQ (amino acids 1 to 450) with a gentamicin resistance cassette from pPS856 (8), subcloning into pEX100T (9), and delivering it to recipient cells via triparental mating as described previously (10). This derivative (heretofore referred to as ⌬RTQ), along with the parental strain and isogenic pyoverdine biosynthesis-deficient pvdA deletion mutant, was tested in the MB-1 adaptation assay using methods described previously (2).…”

mentioning

confidence: 99%

Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

Tomaras

Crandon

McPherson

et al. 2015

Self Cite

Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability.T he extent and severity of antibiotic resistance in Gram-negative pathogens require the development of innovative therapeutic interventions that not only circumvent existing clinically relevant resistance mechanisms but will also result in limited spontaneous resistance emergence. Although standard in vitro methodologies exist for predicting resistance development, translational deficiencies that can stem from their lack of in vivo physiological relevance are becoming increasingly apparent. We previously described the siderophore-monobactam conjugate MB-1 ( Fig. 1 [1]), which has broad-spectrum in vitro activities against multidrug-resistant (MDR) Gram-negative pathogens, including Pseudomonas aeruginosa, when tested using standard methods established by the Clinical and Laboratory Standards Institute (CLSI) (1). Unfortunately, we have also demonstrated that despite these encouraging in vitro activities, the efficacy of MB-1 in a neutropenic murine infection model was highly variable and not directly predictable from traditional in vitro activity assays (2). Instead, the development of a modified in vitro resistance frequency assay, which relies on the utilization of more in vivo-relevant iron conditions, has proven to be more predictive of the efficacy of MB-1 in vivo. Additionally, this assay has demonstrated a role for endogenous siderophores, such as pyoverdine, in mediating the adaptive cellular response that enables P. aeruginosa to transiently resist MB-1 activity (2). While these results suggest that the use of MB-1 as monotherapy is not a viable option, we have speculated about alternative strategies that may mitigate this adaptive response and resurrect MB-1 as a legitimate antibacterial agent. Among these potentiation-type approaches, we considered the idea that inhibiting endogenous siderophore functionality should circumvent the adaptation phenotype mediated by the competition for iron by native siderophore systems. Siderophore secretion into the extracellular milieu has been shown to be dependent on functional efflux pumps in Gram-negative bacteria (3-5). In the case of P. aeruginosa, the secretion of newly synthesized pyoverdine is accomplished by the ABC transporter-type efflux system encoded by pvdR, pvdT, and opmQ (6). Additionally, this system is responsible for pyoverdine recycling after successive rounds of iron acquisition and delivery (7).To prove that inhibiting the activity of this pump can potentiate the activity of MB-1, we generated a pvdRT-op...