ABSTRACT
Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance.
Subject(s)
Ampicillin Resistance/genetics , Ampicillin/pharmacology , DNA, Bacterial/genetics , Enterococcus faecium/genetics , Gene Expression Regulation, Bacterial , Penicillin-Binding Proteins/genetics , Anti-Bacterial Agents/pharmacology , Chromosome Mapping , DNA, Bacterial/metabolism , Enterococcus faecium/classification , Enterococcus faecium/drug effects , Enterococcus faecium/isolation & purification , Genetic Variation , Genotype , Gram-Positive Bacterial Infections/microbiology , Humans , Microbial Sensitivity Tests , Penicillin-Binding Proteins/metabolism , Phenotype , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The classic nitrobenzene anion-radical (NB(-*) or nitrobenzenide) is isolated for the first time as pure crystalline alkali-metal salts. The deliberate use of the supporting ligands 18-crown-6 and [2.2.2]cryptand allows the selective formation of contact ion pairs designated as (crown)M(+)NB(-*), where M(+) = K(+), Rb(+), and Cs(+), as well as the separated ion pair K(cryptand)(+)NB(-*)-both series of which are structurally characterized by precise low-temperature X-ray crystallography, ESR analysis, and UV-vis spectroscopy. The unusually delocalized structure of NB(-*) in the separated ion pair follows from the drastically shortened N-C bond and marked quinonoidal distortion of the benzenoid ring to signify complete (95%) electronic conjugation with the nitro substituent. On the other hand, the formation of contact ion pairs results in the substantial decrease of electronic conjugation in inverse order with cation size (K(+) > Rb(+)) owing to increased localization of negative charge from partial (NO(2)) bonding to the alkali-metal cation. Such a loss in electronic conjugation (or reverse charge transfer) may be counterintuitive, but it is in agreement with the distribution of odd-electron spin electron density from the ESR data and with the hypsochromic shift of the characteristic absorption band in the electronic spectra. Most importantly, this crystallographic study underscores the importance of ion-pair structure on the intrinsic property (and thus reactivity) of the component ions-as focused here on the nitrobenzenide anion.
