Project description:The bacterial pathogen, Acinetobacter baumannii, is a leading cause of drug-resistant infections. Here, we investigated the potential of developing nanobodies that specifically recognize A. baumannii over other Gram-negative bacteria. Through generation and panning of a synthetic nanobody library, we identified several potential lead candidates. We demonstrate how incorporation of next generation sequencing analysis can aid in selection of lead candidates for further characterization. Using monoclonal phage display, we validated the binding of several lead nanobodies to A. baumannii. Subsequent purification and biochemical characterization revealed one particularly robust nanobody that broadly and specifically bound A. baumannii compared to other common drug resistant pathogens. These findings support the potentially for nanobodies to selectively target A. baumannii and the identification of lead candidates for possible future diagnostic and therapeutic development.
Project description:In recent years, the Gram-negative bacterium Acinetobacter baumannii has garnered considerable attention for its unprecedented capacity to rapidly develop resistance to antibacterial therapeutics. This is coupled with the seemingly epidemic emergence of new hyper-virulent strains. Although strain-specific differences for A. baumannii isolates have been well described, these studies have primarily focused on proteinaceous factors. At present, only limited publications have investigated the presence and role of small regulatory RNA (sRNA) transcripts. Herein, we perform such an analysis, describing the RNA-seq-based identification of 78 A. baumannii sRNAs in the AB5075 background. Together with six previously identified elements, we include each of these in a new genome annotation file, which will serve as a tool to investigate regulatory events in this organism. Our work reveals that the sRNAs display high expression, accounting for >50 % of the 20 most strongly expressed genes. Through conservation analysis we identified six classes of similar sRNAs, with one found to be particularly abundant and homologous to regulatory, C4 antisense RNAs found in bacteriophages. These elements appear to be processed from larger transcripts in an analogous manner to the phage C4 molecule and are putatively controlled by two further sRNAs that are strongly antisense to them. Collectively, this study offers a detailed view of the sRNA content of A. baumannii, exposing sequence and structural conservation amongst these elements, and provides novel insight into the potential evolution, and role, of these understudied regulatory molecules. This study is based on the annotation of novel sRNAs on basis of an Acinetobacter baumannii RNA sequencing dataset. Each sample was generated by pooling three independent biological replicate RNA preps
Project description:LpxC and lpxD are involved in the synthesis of bacterial LPS and are essential for the maintenance of bacterial outer membrane integrity.Here, we show that loss of lpxC and lpxD affects energy metabolism in A. baumannii and is associated with bacterial-phage interactions.The lpxC and lpxD deficient strains showed significantly different changes compared with the parental strain under phage pressure.
Project description:Acinetobacter baumannii is currently a major threat to human health. With the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, the development of complementary strategies is needed. A promising complimentary and realistic strategy could be phage therapy, which uses bacteriophages (phages), i.e viruses that specifically infect and kill bacterial cells during their life cycle. We designed a two-phage cocktail highly efficient against an extensive drug-resistant (XDR) A. baumannii isolate collected from a patient with burn wound infection at CHUV (termed Ab125). A first in vitro screen of our collection of 34 different phages identified only phage vB_AbaM_3098 as capable of lysing Ab125. However, quick selection of phage-resistant clones (termed Ab139) occurred. Comparative genomics and proteomics between Ab125 and Ab139 revealed several key variations. Very interestingly, we observed that Ab139 became susceptible to six different phages in the collection, otherwise inactive on Ab125. Phage-resistance was also selected when Ab139 was challenged with either of the six phages, with bacterial regrowth observed between 14 h and 16 h. However, combination of vB_AbaM_3098 and vB_AbaM_3014 led to a two-phage cocktail capable of totally inhibiting the growth of Ab125. Treatment with the phage cocktail led to 90% survival after 5 days in the in vivo Galleria Mellonella model of infectious diseases, compared to 0% in the non-treated group. We show that the combination of a phage that only slightly shifted the in vitro bacterial growth curve with an “inactive phage” led to the formulation of a highly bactericidal phage cocktail against Ab125. We then tested the therapeutic potential of the assembled cocktail in synergy with antibiotics and found a synergy with colistin. This work highlights the complexity sometimes involved in the assembly of potent phage cocktail.
Project description:A major reservoir for spread of the emerging pathogen Acinetobacter baumannii is hopsital surfaces, where bacteria persist in a desiccated state. To identify gene products influencing desiccation survival, a transposon sequencing (Tn-seq) screen was performed. Using this approach, we identified genes both positively and negatively impacting the desiccation tolerance of A. baumannii.