Fabrizia Minandri

In Vitro and In Vivo Antimicrobial Activities of Gallium Nitrate against Multidrug-Resistant Acinetobacter baumannii

ABSTRACT Multidrug-resistant Acinetobacter baumannii poses a tremendous challenge to traditional antibiotic therapy. Due to the crucial role of iron in bacterial physiology and pathogenicity, we investigated iron metabolism as a possible target for anti-A. baumannii chemotherapy using gallium as an iron mimetic. Due to chemical similarity, gallium competes with iron for binding to several redox enzymes, thereby interfering with a number of essential biological reactions. We found that Ga(NO3)3, the active component of an FDA-approved drug (Ganite), inhibits the growth of a collection of 58 A. baumannii strains in both chemically defined medium and human serum, at concentrations ranging from 2 to 80 μM and from 4 to 64 μM, respectively. Ga(NO3)3 delayed the entry of A. baumannii into the exponential phase and drastically reduced bacterial growth rates. Ga(NO3)3 activity was strongly dependent on iron availability in the culture medium, though the mechanism of growth inhibition was independent of dysregulation of gene expression controlled by the ferric uptake regulator Fur. Ga(NO3)3 also protected Galleria mellonella larvae from lethal A. baumannii infection, with survival rates of ≥75%. At therapeutic concentrations for humans (28 μM plasma levels), Ga(NO3)3 inhibited the growth in human serum of 76% of the multidrug-resistant A. baumannii isolates tested by ≥90%, raising expectations on the therapeutic potential of gallium for the treatment of A. baumannii bloodstream infections. Ga(NO3)3 also showed strong synergism with colistin, suggesting that a colistin-gallium combination holds promise as a last-resort therapy for infections caused by pan-resistant A. baumannii.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

ABSTRACT Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/.

Evidence of Diversity among Epidemiologically Related Carbapenemase-Producing Acinetobacter baumannii Strains Belonging to International Clonal Lineage II

ABSTRACT Carbapenem-resistant Acinetobacter baumannii strains belonging to international clonal lineage II (ICL-II) have become predominant in intensive care units (ICUs) throughout Italy. Between 2005 and 2009, the carbapenem-hydrolyzing class D β-lactamase (CHDL) bla OXA-23 gene became more prevalent than bla OXA-58 among epidemic ICL-II strains showing extensive genetic similarity. These findings posed the question of whether CHDL gene replacement occurred in the homogeneous ICL-II population or a new OXA-23 clone(s) emerged and spread in ICUs. In this study, the changes in the ICL-II A. baumannii population and CHDL gene carriage were investigated in 30 genetically related isolates collected during the bla OXA-58-to-bla OXA-23 transition period. Pulsotyping, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST) results were combined with multilocus variable-number tandem-repeat (VNTR) analysis (MLVA-8), siderotyping, and plasmid profiling to improve genotype-based discrimination between isolates. Pulsotyping, RAPD analysis, and MLST clustered isolates into a single type. MLVA-8 identified 19 types that clustered into three complexes. All OXA-23-producing isolates formed a single complex, while OXA-58 producers were split into two complexes. Southern blot analysis of the physical localization and genetic context of the CHDL genes showed that bla OXA-58 was invariably located on plasmids, while bla OXA-23 was present within Tn2006 on the chromosome or both the chromosome and plasmids. These data indicate that the apparently homogeneous population of CHDL-producing ICL-II strains was composed of several independent strains and that, between 2005 and 2009, distinct OXA-23 producers displaced the preexisting OXA-58 producers. Thus, MLVA-8 appears to be a suitable tool not only for investigating A. baumannii population structure but also for high-resolution epidemiological typing.

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

ABSTRACT Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities.

Repurposing of gallium-based drugs for antibacterial therapy

While the occurrence and spread of antibiotic resistance in bacterial pathogens is vanishing current anti‐infective therapies, the antibiotic discovery pipeline is drying up. In the last years, the repurposing of existing drugs for new clinical applications has become a major research area in drug discovery, also in the field of anti‐infectives. This review discusses the potential of repurposing previously approved gallium formulations in antibacterial chemotherapy. Gallium has no proven function in biological systems, but it can act as an iron‐mimetic in both prokaryotic and eukaryotic cells. The activity of gallium mostly relies on its ability to replace iron in redox enzymes, thus impairing their function and ultimately hampering cell growth. Cancer cells and bacteria are preferential gallium targets due to their active metabolism and fast growth. The wealth of knowledge on the pharmacological properties of gallium has opened the door to the repurposing of gallium‐based drugs for the treatment of infections sustained by antibiotic‐resistant bacterial pathogens, such as Acinetobacter baumannii or Pseudomonas aeruginosa, and for suppression of Mycobacterium tuberculosis growth. The promising antibacterial activity of gallium both in vitro and in different animal models of infection raises the hope that gallium will confirm its efficacy in clinical trials, and will become a valuable therapeutic option to cure otherwise untreatable bacterial infections.

Promises and failures of gallium as an antibacterial agent.

Gallium has a long history as a diagnostic and chemotherapeutic agent. The pharmacological properties of Ga(III) rely on chemical mimicry; when Ga(III) is exogenously supplied to living cells it can replace Fe(III) within target molecules, thereby perturbing bacterial metabolism. Ga(III)-induced metabolic distresses are dramatic in fast-growing cells, like bacterial cells. Interest in the antibacterial properties of Ga(III) has been raised by the compelling need for novel drugs to combat multidrug-resistant bacteria and by the shortage of new antibiotic candidates in the pharmaceutical pipeline. Ga(III) activity has been demonstrated, both in vitro and in animal models of infections, on several bacterial pathogens, also including intracellular and biofilm-forming bacteria. Ga(III) activity is affected by iron availability and the metabolic state of the cell, being maximal in iron-poor media and in respiring cells. Synergism between Ga(III) and antibiotics holds promise as last resort therapy for infections sustained by pandrug-resistant bacteria.

Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa

ABSTRACT Gallium (Ga) is an iron mimetic that has successfully been repurposed for antibacterial chemotherapy. To improve the antibacterial potency of Ga on Pseudomonas aeruginosa, the effect of complexation with a variety of siderophores and synthetic chelators was tested. Ga complexed with the pyochelin siderophore (at a 1:2 ratio) was more efficient than Ga(NO3)3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga entrance into the cell through the pyochelin translocon.

The Dual Personality of Iron Chelators: Growth Inhibitors or Promoters?

Iron is an essential nutrient for almost all living cells, and pathogenic bacteria are faced with severe iron limitation in the mammalian host ([1][1]). To make iron available during infection, most microorganisms produce and transport into the cell specific iron chelators (siderophores) or acquire

Iron and Acinetobacter baumannii Biofilm Formation

Acinetobacter baumannii is an emerging nosocomial pathogen, responsible for infection outbreaks worldwide. The pathogenicity of this bacterium is mainly due to its multidrug-resistance and ability to form biofilm on abiotic surfaces, which facilitate long-term persistence in the hospital setting. Given the crucial role of iron in A. baumannii nutrition and pathogenicity, iron metabolism has been considered as a possible target for chelation-based antibacterial chemotherapy. In this study, we investigated the effect of iron restriction on A. baumannii growth and biofilm formation using different iron chelators and culture conditions. We report substantial inter-strain variability and growth medium-dependence for biofilm formation by A. baumannii isolates from veterinary and clinical sources. Neither planktonic nor biofilm growth of A. baumannii was affected by exogenous chelators. Biofilm formation was either stimulated by iron or not responsive to iron in the majority of isolates tested, indicating that iron starvation is not sensed as an overall biofilm-inducing stimulus by A. baumannii. The impressive iron withholding capacity of this bacterium should be taken into account for future development of chelation-based antimicrobial and anti-biofilm therapies.