Cefiderocol (CFDC; S-649266), a novel parenteral siderophore cephalosporin conjugated with a catechol moiety, has a characteristic antibacterial spectrum with a potent activity against a broad range of aerobic Gram-negative bacterial species, including carbapenem-resistant strains of Enterobacteriaceae and nonfermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii. Cefiderocol has affinity mainly for penicillin-binding protein 3 (PBP3) of Enterobacteriaceae and nonfermenting bacteria similar to that of ceftazidime. A deficiency of the iron transporter PiuA in P. aeruginosa or both CirA and Fiu in Escherichia coli caused 16-fold increases in cefiderocol MICs, suggesting that these iron transporters contribute to the permeation of cefiderocol across the outer membrane. The deficiency of OmpK35/36 in Klebsiella pneumoniae and the overproduction of efflux pump MexA-MexB-OprM in P. aeruginosa showed no significant impact on the activity of cefiderocol.

Cefiderocol (S-649266) is a novel parenteral siderophore cephalosporin conjugated with a catechol moiety at the third-position side chain. The in vitro activity of cefiderocol against Pseudomonas aeruginosa was enhanced under iron-depleted conditions, whereas that of ceftazidime was not affected. The monitoring of [thiazole-14C]cefiderocol revealed the increased intracellular accumulation of cefiderocol in P. aeruginosa cells incubated under iron-depleted conditions compared with those incubated under iron-sufficient conditions. Cefiderocol was shown to have potent chelating activity with ferric iron, and extracellular iron was efficiently transported into P. aeruginosa cells in the presence of cefiderocol as well as siderophores, while enhanced transport of extracellular ferric iron was not observed when one of the hydroxyl groups of the catechol moiety of cefiderocol was replaced with a methoxy group. We conclude that cefiderocol forms a chelating complex with iron, which is actively transported into P. aeruginosa cells via iron transporters, resulting in potent antibacterial activity of cefiderocol against P. aeruginosa.

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 g/ml), kanamycin (25 g/ml), and ofloxacin (10 g/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.Reduced susceptibility of biofilm bacteria to antimicrobial agents is a crucial problem for treatment of chronic infections (11,29,48). It has been estimated that 65% of microbial infections are associated with biofilms (11,29,37), and biofilm cells are 100 to 1,000 times more resistant to antimicrobial agents than planktonic bacterial cells (11,29,32).The molecular nature of this apparent resistance has not been elucidated well, and a number of mechanisms have been proposed to explain the reduced susceptibility, such as restricted antibiotic penetration (47), decreased growth rates and metabolism (7, 52), quorum sensing and induction of a biofilmspecific phenotype (8,29,35,39,49), stress response activation (7,52), and an increase in expression of efflux...

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.

S-649266 is a novel antibiotic with potent in vitro activity against a range of non-fermenting Gram-negative bacteria, including MDR strains.

cTo better understand the antibacterial activity of S-649266 against carbapenemase producers, its stability against clinically relevant carbapenemases was investigated. The catalytic efficiencies (k cat /K m ) of IMP-1, VIM-2, and L1 for S-649266 were 0.0048, 0.0050, and 0.024 M ؊1 s ؊1 , respectively, which were more than 260-fold lower than that for meropenem. Only slight hydrolysis of S-649266 against KPC-3 was observed. NDM-1 hydrolyzed meropenem 3-fold faster than S-649266 at 200 M.

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.

Biofilms gain resistance to various antimicrobial agents, and the presence of antibiotic resistance genes is thought to contribute to a biofilm-mediated antibiotic resistance. Here we showed the interplay between the tetracycline resistance efflux pump TetA(C) and the ampicillin resistance gene (bla TEM-1 ) in biofilms of Escherichia coli harboring pBR322 in the presence of the mixture of ampicillin and tetracycline. E. coli in the biofilms could obtain the high-level resistance to ampicillin, tetracycline, penicillin, erythromycin, and chloramphenicol during biofilm development and maturation as a result of the interplay between the marker genes on the plasmids, the increase of plasmid copy number, and consequently the induction of the efflux systems on the bacterial chromosome, especially the EmrY/K and EvgA/S pumps. In addition, we characterized the overexpression of the TetA(C) pump that contributed to osmotic stress response and was involved in the induction of capsular colanic acid production, promoting formation of mature biofilms. However, this investigated phenomenon was highly dependent on the addition of the subinhibitory concentrations of antibiotic mixture, and the biofilm resistance behavior was limited to aminoglycoside antibiotics. Thus, marker genes on plasmids played an important role in both resistance of biofilm cells to antibiotics and in formation of mature biofilms, as they could trigger specific chromosomal resistance mechanisms to confer a high-level resistance during biofilm formation.