Luis Actis, Miami University, Oxford, Ohio, USA
Responses of Acinetobacter baumannii to extracellular signals that affect virulence and host-pathogen interactions
Acinetobacter baumannii is recognized by its capacity to adapt to and persist in different environments by sensing and responding to a wide range of extracellular signals including iron limitation imposed by the host. This signal controls the expression of critical iron metabolic and acquisition functions, mainly via the production and utilization of high-affinity siderophores, which could be negatively affected by the non-ferric metalloporphyrin derivative Ga-PPIX. Iron limitation also regulates the expression of functions that could supply iron from host sources such as the production of iron-regulated phospholipases, which could be inhibited by the competitive inhibitor miltefosine and therefore significantly affect the virulence of this pathogen. Sensing and responding to abiotic and biotic surfaces via the BfmRS two-component regulatory system has been recognized as an A. baumannii critical virulence factor. This transcriptional regulatory system controls the differential expression of genes coding for a wide range of functions including the interaction with abiotic and biotic surfaces, biofilm and pili production, surface motility as well as important cellular function such as the expression of genes coding for central metabolism and the unexpected differential expression of siderophore-mediated iron acquisition and utilization functions. The presence of mucin also proved to be an important biotic signal A. baumannii responds to by modulating the expression of genes involved in host pathogen interactions and virulence, including the differential expression of a type 6 secretion system and genes involved in phenylacetic acid metabolism. Altogether, these observations show that A. baumannii is capable to adapt to different environmental conditions by sensing and globally responding to environmental and host conditions that allow this pathogen to persist in medical settings and cause infections in compromised hosts.
Patrice Courvalin, Institut Pasteur, Paris, France
Multidrug resistance by efflux in Acinetobacter
In addition to its capacity to acquire genetic determinants, overproduction of RND efflux pumps with broad substrate specificity has been shown to be associated with multidrugresistance (MDR) in A. baumannii. To date, three Acinetobacter Drug Efflux (Ade) RND systems, AdeABC, AdeFGH, and AdeIJK have been characterized in A. baumannii. Overexpression of AdeABC, primarily, and of AdeFGH play a major role in acquired resistance whereas AdeIJK contributes to intrinsic resistance. Expression of each pump is tightly regulated by a different mechanism: AdeABC by a two-component regulatory system, AdeRS, AdeFGH by the LysR-type transcriptional regulator AdeL, and AdeIJK by the TetR transcriptional regulator AdeN. To characterize the contribution of AdeABC, AdeFGH, and AdeIJK efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii, a set of isogenic mutants 1) overexpressing each system following a point mutation in their cognate regulator or 2) deleted for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquire antibiotic resistance when overproduced. AdeABC has a broad substrate range including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol. and it confers clinical resistance to aminoglycosides. When combined with enzymatic resistance to carbapenems and aminoglycosides this pump contributes in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK is expressed constitutively and responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. The study of the relative contribution of these three most prevalent pumps in 14 clinical isolates, distinct on the basis of MLST and PFGE, outlined the high incidence of adeABC overexpression in MDR strains, resulting from a variety of point mutations in AdeRS, whereas only half of the strains had a slight increase in adeFGH expression and, curiously, none overexpressed adeIJK.
Emmanuelle Dé, Université de Rouen, Mont Saint Aignan, France
Growth of Acinetobacter baumannii at the air-liquid interface: pellicle as a full lifestyle
Many bacteria, among which A. baumannii, have the ability to colonize the upper surface of static liquids, forming a biofilm at the air-liquid interface named “pellicle”. The Air-liquid Interface (ALI) is a favourable niche for strictly aerobic bacteria which can obtain oxygen from the air and nutrients from the liquid media. Despite the ubiquity of these pellicles in both natural and artificial environments, few studies have investigated this biofilm type. We performed the global dynamic study of the whole proteome of A. baumannii ATCC 17978 grown as pellicle, using a proteomic quantitative approach. Results accord with the general findings reporting that sessile bacteria are far more resistant to detrimental conditions than their planktonic counterparts, but also suggested a correlation between the pellicle forming ability and the bacterial virulence. Indeed, we showed the accumulation of numerous virulence factors during the pellicle growth, e.g., phospholipases, adhesion factors or Type 6 Secretion System (T6SS) as well as some regulators like GacAS two-component system. We also highlighted that the bacterial adaptation to this growth mode requires drastic changes at the membrane level: Bam and Tam systems, both related to the OM insertion machinery, play thus a critical role during pellicle biogenesis. Acetylation/deacetylation post-translational modifications may also participate to the regulation network of this A. baumannii lifestyle.
Ellen Neidle, University of Georgia, Athens, USA
EASy does it: a new method for Acinetobacter research
A new method for experimental evolution has been developed and dubbed Evolution by Amplification and Synthetic Biology (EASy). Using the facile genetic system of Acinetobacter baylyi ADP1, desired mutants were rapidly generated after the creation of tandem arrays of chromosomal segments. Increased gene dosage promotes the evolution of traits, not otherwise possible, by conferring new phenotypes. Under selection, gene copies diverge and segregate. This method was demonstrated using a catabolic pathway in which foreign genes were inserted into the chromosome, amplified, and allowed to evolve under selective growth conditions. Variant enzymes, derived from the foreign DNA, were selected that were functional in A. baylyi and in Pseudomonas putida. Additionally, some chromosomal mutations were selected that were outside the region of amplified DNA. These mutations were in a gene that appears to affect carbon source utilization. The homologous region of Acinetobacter baumannii has been predicted to play a role in pathogenesis. These studies suggest that EASy is a powerful method to investigate the similarities and differences between A. baylyi and A. baumannii for a variety of physiological and pathogenic topics.
Jordi Vila Estape, Hospital Clinic, Barcelona, Spain
Current and future diagnostic and therapeutic options
Acinetobacter species have emerged as one of the high-priority hospital-acquired pathogens causing substantial morbidity and mortality. Currently, the Acinetobacter baumannii group is constituted by the following species: Acinetobacter baumannii, A. nosocomialis, Acinetobacter seifertii,
Nowadays, the increased resistance of A. baumannii to most antibacterial agents including colistin make it necessary to discover new compounds active against pandrug resistant strains. Among 30 different derivatives of ciprofloxacin, one (UB8901) showed good activity (MIC of 0.5 mg/L) against A. baumannii with a mutation in the gyrA gene, whereas the MIC of ciprofloxacin for the same strain was 8 mg/L. It showed a good pharmacokinetic parameters and in vivo activity. We also tested the activity of 14 peptides against colistin-susceptible and colistin-resistant A. baumannii clinical isolates; mastoparan showed a MIC of 4 and 1 mg/L for colistin-susceptible and colistin-resistant strains. We optimized this peptide in order to increase its stability in serum up to 24 hours.
Ceragenins are compounds generated from cholic acid, named peptidomimetics due to their amphipathicity similar to antimicrobial peptides. We investigated the activity of these compounds against multidrug-resistant A. baumannii. Four different ceragenins (CSA-138, CSA-131, CSA-44 and CSA-13) were tested against both colistin-susceptible and colistin-resistant A. baumannii strains using a microdilution method. Ceragenins were also tested against 15 strains of epidemiologically-unrelated A. baumannii in order to check the previous results. The MICs of the ceragenins against both colistin-susceptible and –resistant A. baumannii ranged from 2 to 8 mg/L. Killing curves of the most active compound, CSA-131, were performed against colistin-resistant A. baumannii, showing a bactericidal effect at all the concentrations at 4h. However, regrowth was observed at lower concentrations (MIC, 2xMIC, 4xMIC).
Finally, preventing the adhesion of pathogens to host cells provides an additional approach to tackling multidrug-resistant bacteria. In this regard, the identification of OmpA as a key bacterial virulence factor has been a major breakthrough. Using virtual screening based on knowledge of the target, we identified a cyclic hexapeptide AOA-2 that inhibits the adhesion of A. baumannii to host cells, thereby preventing the development of infection in vitro. AOA-2 does not inhibit bacterial growth. Inhibition of OmpA offers a strategy to address the urgent need for treatments for infections caused by A. baumannii.
Paolo Visca, University Roma Tre, Rome, Italy
Antibacterial strategies targeting Acinetobacter baumannii iron metabolism
Treatment of Acinetobacter baumannii infection is problematic due to the high frequency of multi-drug resistant (MDR) isolates. The crucial role of iron in bacterial infection and pathogenicity has paved the way for the development of antibacterial strategies targeting iron metabolism. Gallium is an iron-mimetic metal which inhibits bacterial growth by interfering with a number of iron-dependent biological functions. We initially investigated the in vitro activity of gallium (gallium nitrate, GaN; gallium dicitrate, Ga-Cit2) on a panel of A. baumannii strains, including MDR isolates. We found that gallium activity strongly depends on iron availability in the culture medium, and GaN is more active in the iron-poor medium M9 or in complement-free human serum (HS) than in other media (e.g., Mueller Hinton, MH or Dialyzed Trypticase Soy Broth, TSBD). The concentration of GaN required to inhibit A. baumannii growth ranged from 2 to 80 μM in M9 and 4 to 64 μM in HS, while it was higher in TSBD or MH (>256 μM). GaN also protected Galleria mellonella larvae from lethal A. baumannii infection (> 75% of survival) and showed synergism with colistin, raising hope for combination therapy against A. baumannii infections.
We also investigated the effect of GaN on A. baumannii biofilm formation. We observed that TSBD was a suitable medium to study the development of A. baumannii biofilm, but GaN activity in TSBD was extremely poor. We also provide evidence that both prototypic and clinical A. baumannii strains can develop as mature biofilms in HS. We characterized both the development and the architecture of A. baumannii biofilm in HS (e.g. biomass, thickness and volume) through confocal microscopy and image analysis. We demonstrated that 16 μM GaN drastically reduces A. baumannii biofilm formation in HS, whereas 64 μM GaN causes massive disruption of pre-formed A. baumannii biofilm. Since indwelling medical devices are primary colonization sites for A. baumannii dissemination, these findings point to a possible use of gallium as an anti-A. baumannii agent for the coating of biomaterials.