The Iron Content of Human Serum Albumin Modulates the Susceptibility of Acinetobacter baumannii to Cefiderocol.

The mortality rates of patients infected with Acinetobacter baumannii who were treated with cefiderocol (CFDC) were not as favorable as those receiving the best available treatment for pulmonary and bloodstream infections. Previous studies showed that the presence of human serum albumin (HSA) or HSA-containing fluids, such as human serum (HS) or human pleural fluid (HPF), in the growth medium is correlated with a decrease in the expression of genes associated with high-affinity siderophore-mediated iron uptake systems. These observations may explain the complexities of the observed clinical performance of CFDC in pulmonary and bloodstream infections, because ferric siderophore transporters enhance the penetration of CFDC into the bacterial cell. The removal of HSA from HS or HPF resulted in a reduction in the minimal inhibitory concentration (MIC) of CFDC. Concomitant with these results, an enhancement in the expression of TonB-dependent transporters known to play a crucial role in transporting iron was observed. In addition to inducing modifications in iron-uptake gene expression, the removal of HSA also decreased the expression of ß-lactamases genes. Taken together, these observations suggest that environmental HSA has a role in the expression levels of select A. baumannii genes. Furthermore, the removal of iron from HSA had the same effect as the removal of HSA upon the expression of genes associated with iron uptake systems, also suggesting that at least one of the mechanisms by which HSA regulates the expression of certain genes is through acting as an iron source.

Human Serum Proteins and Susceptibility of Acinetobacter baumannii to Cefiderocol: Role of Iron Transport.

Cefiderocol, a recently introduced antibiotic, has a chemical structure that includes a cephalosporin that targets cell wall synthesis and a chlorocatechol siderophore moiety that facilitates cell penetration by active iron transporters. Analysis of the effect that human serum, human serum albumin, and human pleural fluid had on growing Acinetobacter baumannii showed that genes related to iron uptake were down-regulated. At the same time, ß-lactamase genes were expressed at higher levels. The minimum inhibitory concentrations of this antimicrobial in A. baumannii cells growing in the presence of human serum, human serum albumin, or human pleural fluid were higher than those measured when these fluids were absent from the culture medium. These results correlate with increased expression levels of ß-lactamase genes and the down-regulation of iron uptake-related genes in cultures containing human serum, human serum albumin, or human pleural fluid. These modifications in gene expression could explain the less-than-ideal clinical response observed in patients with pulmonary or bloodstream A. baumannii infections. The exposure of the infecting cells to the host's fluids could cause reduced cefiderocol transport capabilities and increased resistance to ß-lactams. The regulation of genes that could impact the A. baumannii susceptibility to cefiderocol, or other antibacterials, is an understudied phenomenon that merits further investigation.

Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility.

Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res 68:5318-5325, 2008) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.

Silencing Antibiotic Resistance with Antisense Oligonucleotides.

Antisense technologies consist of the utilization of oligonucleotides or oligonucleotide analogs to interfere with undesirable biological processes, commonly through inhibition of expression of selected genes. This field holds a lot of promise for the treatment of a very diverse group of diseases including viral and bacterial infections, genetic disorders, and cancer. To date, drugs approved for utilization in clinics or in clinical trials target diseases other than bacterial infections. Although several groups and companies are working on different strategies, the application of antisense technologies to prokaryotes still lags with respect to those that target other human diseases. In those cases where the focus is on bacterial pathogens, a subset of the research is dedicated to produce antisense compounds that silence or reduce expression of antibiotic resistance genes. Therefore, these compounds will be adjuvants administered with the antibiotic to which they reduce resistance levels. A varied group of oligonucleotide analogs like phosphorothioate or phosphorodiamidate morpholino residues, as well as peptide nucleic acids, locked nucleic acids and bridge nucleic acids, the latter two in gapmer configuration, have been utilized to reduce resistance levels. The major mechanisms of inhibition include eliciting cleavage of the target mRNA by the host's RNase H or RNase P, and steric hindrance. The different approaches targeting resistance to ß-lactams include carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones. The purpose of this short review is to summarize the attempts to develop antisense compounds that inhibit expression of resistance to antibiotics.

Bridged Nucleic Acids Reloaded.

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.

Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia.

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.

Inhibition of AAC(6')-Ib-mediated resistance to amikacin in Acinetobacter baumannii by an antisense peptide-conjugated 2',4'-bridged nucleic acid-NC-DNA hybrid oligomer.

Multiresistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6')-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6')-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro. An isosequential nuclease-resistant hybrid oligomer composed of 2',4'-bridged nucleic acid-NC (BNA(NC)) residues and deoxynucleotides (BNA(NC)-DNA) conjugated to the permeabilizing peptide (RXR)4XB ("X" and "B" stand for 6-aminohexanoic acid and ß-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6')-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls.

Aminoglycoside modifying enzymes.

Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different -OH or -NH2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltransferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes.

Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum.

Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa predicted protein, AngN. In this work we show that AngN is essential for anguibactin biosynthesis and possesses two domains with homology to cyclization (Cy) domains of NRPSs. Substitution by alanine of the aspartic acid residues within a conserved motif of either Cy1 or Cy2 domain demonstrated the importance of these two domains in AngN function during siderophore biosynthesis. Site-directed mutations in both domains (D133A/D575A and D138A/D580A) resulted in anguibactin-deficient phenotypes while mutations in each domain did not abolish siderophore production but caused a reduction in the amounts produced. The mutations D133A/D575A and D138A/D580A also resulted as expected in a dramatic attenuation of the virulence of V. anguillarum 775 highlighting the importance of this gene for the biosynthesis of anguibactin within the vertebrate host. Regulation of the angN gene follows the patterns observed at the iron transport-biosynthesis promoter with angN transcription repressed in the presence of iron and enhanced by AngR and trans-acting factor (TAF) under iron limitation.

The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related.

The Acinetobacter baumannii type strain, ATCC 19606, secretes acinetobactin, a catechol siderophore highly related to the iron chelator anguibactin produced by the fish pathogen Vibrio anguillarum (Listonella anguillarum). This paper reports the initial characterization of the genes and gene products involved in the acinetobactin-mediated iron-acquisition process. Insertional mutagenesis resulted in the isolation of several derivatives whose ability to grow in medium containing the iron chelator 2,2'-dipyridyl was affected. One of the insertions disrupted a gene encoding a predicted outer-membrane protein, named BauA, highly similar to FatA, the receptor for ferric anguibactin. Immunological relatedness of BauA with FatA was confirmed by Western blot analysis. Another transposon insertion was mapped to a gene encoding a protein highly similar to FatD, the permease component of the anguibactin transport system. Further DNA sequencing and nucleotide sequence analysis revealed that these A. baumannii 19606 genes are part of a polycistronic locus that contains the bauDCEBA ORFs. While the translation products of bauD, -C, -B and -A are highly related to the V. anguillarum FatDCBA iron-transport proteins, the product of bauE is related to the ATPase component of Gram-positive ATP-binding cassette (ABC) transport systems. This entire locus is flanked by genes encoding predicted proteins related to AngU and AngN, V. anguillarum proteins required for the biosynthesis of anguibactin. These protein similarities, as well as the structural similarity of anguibactin and acinetobactin, suggested that these two siderophores could be utilized by both bacterial strains, a possibility that was confirmed by siderophore utilization bioassays. Taken together, these results demonstrate that these pathogens, which cause serious infections in unrelated hosts, express very similar siderophore-mediated iron-acquisition systems.

A nonribosomal peptide synthetase with a novel domain organization is essential for siderophore biosynthesis in Vibrio anguillarum.

Anguibactin, a siderophore produced by Vibrio anguillarum, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. We have identified a gene from the V. anguillarum plasmid pJM1 that encodes a 78-kDa NRPS protein termed AngM, which is essential in the biosynthesis of anguibactin. The predicted AngM amino acid sequence shows regions of homology to the consensus sequence for the peptidyl carrier protein (PCP) and the condensation (C) domains of NRPSs, and curiously, these two domains are not associated with an adenylation (A) domain. Substitution by alanine of the serine 215 in the PCP domain and of histidine 406 in the C domain of AngM results in an anguibactin-deficient phenotype, underscoring the importance of these two domains in the function of this protein. The mutations in angM that affected anguibactin production also resulted in a dramatic attenuation of the virulence of V. anguillarum 775, highlighting the importance of this gene in the establishment of a septicemic infection in the vertebrate host. Transcription of the angM gene is initiated at an upstream transposase gene promoter that is repressed by the Fur protein in the presence of iron. Analysis of the sequence at this promoter showed that it overlaps the iron transport-biosynthesis promoter and operates in the opposite direction.

Evidence for the active role of a novel nuclease from Helicobacter pylori in the horizontal transfer of genetic information.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach, causes gastritis, and is associated with ulcers and gastric cancer. H. pylori is naturally competent for transformation. Natural genetic transformation is believed to be essential for the genetic plasticity observed in this species. While the relevance of horizontal gene transfer in H. pylori adaptiveness and antibiotic resistance is well documented, the DNA transformation machinery components are barely known. No enzymatic activity associated with the transformation process has been determined experimentally and described. We isolated, microsequenced, and cloned a major DNA nuclease from H. pylori. This protein, encoded by the open reading frame hp0323, was expressed in Escherichia coli. The purified protein, NucT, has a cation-independent thermostable nuclease activity that preferentially cleaves single-stranded DNA. NucT is associated with the membrane. NucT-deficient H. pylori strains are one or more orders of magnitude less efficient than the parental strain for transformation with either chromosomal or self-replicating plasmid DNA. To the best of our knowledge, NucT is the first nuclease identified in a gram-negative natural transformation system, and its existence suggests that there is a mechanism of DNA processing and uptake similar to the mechanisms in well-studied gram-positive systems.

Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775.

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46.5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46.5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.