Susceptibility Testing of Colistin for Acinetobacter baumannii: How Far Are We from the Truth?

Acinetobacter baumannii is involved in life-threatening nosocomial infections, mainly in the intensive care units (ICUs), and often colistin may represent the last therapeutic opportunity. The susceptibility to colistin of 51 epidemiologically typed A. baumannii strains isolated in 2017 from clinical samples of patients hospitalized in the ICU of a tertiary care academic hospital was investigated. All isolates were carbapenem-resistant due to the presence of the bla OXA-23 gene in sequence group 1 (international clonal lineage II) and sequence group 4 (related to international clonal lineage II) isolates, and to the bla OXA-24/40 gene in sequence group 2 (international clonal lineage I) isolates. Vitek®2, agar diffusion, and broth microdilution tests showed major discordancy (≥2 dilution factors) in the minimum inhibitory concentration (MIC) values for colistin in 24 out of 51 isolates, resulting in erroneous reporting of qualitative susceptibility data for eight isolates. In growth kinetics experiments in the presence of colistin, five isolates grew with drug concentrations above the susceptibility breakpoint when incubated for >12 h, and three isolates showed the presence of heteroresistant subpopulations. This study highlights that the high frequency of isolation of carbapenem-resistant A. baumannii strains in high-risk infectious wards requires an accurate application of methods for detecting susceptibility to antibiotics, in particular to colistin, so as to ensure a correct therapeutic approach.

Contribution of Active Iron Uptake to Acinetobacter baumannii Pathogenicity.

Acinetobacter baumannii is an important nosocomial pathogen. Mechanisms that allow A. baumannii to cause human infection are still poorly understood. Iron is an essential nutrient for bacterial growth in vivo, and the multiplicity of iron uptake systems in A. baumannii suggests that iron acquisition contributes to the ability of A. baumannii to cause infection. In Gram-negative bacteria, active transport of ferrisiderophores and heme relies on the conserved TonB-ExbB-ExbD energy-transducing complex, while active uptake of ferrous iron is mediated by the Feo system. The A. baumannii genome invariably contains three tonB genes (tonB1, tonB2, and tonB3), whose role in iron uptake is poorly understood. Here, we generated A. baumannii mutants with knockout mutations in the feo and/or tonB gene. We report that tonB3 is essential for A. baumannii growth under iron-limiting conditions, whereas tonB1, tonB2, and feoB appear to be dispensable for ferric iron uptake. tonB3 deletion resulted in reduced intracellular iron content despite siderophore overproduction, supporting a key role of TonB3 in iron uptake. In contrast to the case for tonB1 and tonB2, the promoters of tonB3 and feo contain functional Fur boxes and are upregulated in iron-poor media. Both TonB3 and Feo systems are required for growth in complement-free human serum and contribute to resistance to the bactericidal activity of normal human serum, but only TonB3 appears to be essential for virulence in insect and mouse models of infection. Our findings highlight a central role of the TonB3 system for A. baumannii pathogenicity. Hence, TonB3 represents a promising target for novel antibacterial therapies and for the generation of attenuated vaccine strains.

New Shuttle Vectors for Gene Cloning and Expression in Multidrug-Resistant Acinetobacter Species.

Understanding bacterial pathogenesis requires adequate genetic tools to assess the role of individual virulence determinants by mutagenesis and complementation assays, as well as for homologous and heterologous expression of cloned genes. Our knowledge of Acinetobacter baumannii pathogenesis has so far been limited by the scarcity of genetic tools to manipulate multidrug-resistant (MDR) epidemic strains, which are responsible for most infections. Here, we report on the construction of new multipurpose shuttle plasmids, namely, pVRL1 and pVRL2, which can efficiently replicate in Acinetobacter spp. and in Escherichia coli The pVRL1 plasmid has been constructed by combining (i) the cryptic plasmid pWH1277 from Acinetobacter calcoaceticus, which provides an origin of replication for Acinetobacter spp.; (ii) a ColE1-like origin of replication; (iii) the gentamicin or zeocin resistance cassette for antibiotic selection; and (iv) a multilinker containing several unique restriction sites. Modification of pVRL1 led to the generation of the pVRL2 plasmid, which allows arabinose-inducible gene transcription with an undetectable basal expression level of cloned genes under uninduced conditions and a high dynamic range of responsiveness to the inducer. Both pVRL1 and pVRL2 can easily be selected in MDR A. baumannii, have a narrow host range and a high copy number, are stably maintained in Acinetobacter spp., and appear to be compatible with indigenous plasmids carried by epidemic strains. Plasmid maintenance is guaranteed by the presence of a toxin-antitoxin system, providing more insights into the mechanism of plasmid stability in Acinetobacter spp.

Acinetobacter baumannii Biofilm Formation in Human Serum and Disruption by Gallium.

Biofilm-associated infections caused by Acinetobacter baumannii are extremely recalcitrant to antibiotic treatment. We report that A. baumannii develops a mature biofilm when grown in complement-free human serum (HS). We demonstrate that 16 µM gallium nitrate (GaN) drastically reduces A. baumannii growth and biofilm formation in HS, whereas 64 µM GaN causes massive disruption of preformed A. baumannii biofilm. These findings pave the way to the repurposing of GaN as an antibiofilm agent for A. baumannii.

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.