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Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

ABSTRACT: Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infection-associated morbidity. Interestingly, this effective cocktail is composed of four phages that do not kill the parent strain of the infection and one phage that simply delays bacterial growth in vitro via a strong but incomplete selection event. The cocktail here appears to function in a combinatorial manner, as one constituent phage targets capsulated A. baumannii bacteria and selects for loss of receptor, shifting the population to an uncapsulated state that is then sensitized to the remaining four phages in the cocktail. Additionally, capsule is a known virulence factor for A. baumannii, and we demonstrated that the emergent uncapsulated bacteria are avirulent in a Galleria mellonella model. These results highlight the importance of anticipating population changes during phage therapy and designing intelligent cocktails to control emergent strains, as well as the benefits of using phages that target virulence factors. Because of the efficacy of this cocktail isolated from a limited environmental pool, we have established a pipeline for developing new phage therapeutics against additional clinically relevant multidrug-resistant pathogens by using environmental phages sourced from around the globe.

SUBMITTER: Regeimbal JM

PROVIDER: S-EPMC5038255 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

Regeimbal James M JM Jacobs Anna C AC Corey Brendan W BW Henry Matthew S MS Thompson Mitchell G MG Pavlicek Rebecca L RL Quinones Javier J Hannah Ryan M RM Ghebremedhin Meron M Crane Nicole J NJ Zurawski Daniel V DV Teneza-Mora Nimfa C NC Biswas Biswajit B Hall Eric R ER

Antimicrobial agents and chemotherapy 20160923 10

Multidrug-resistant bacterial pathogens are an increasing threat to public health, and lytic bacteriophages have reemerged as a potential therapeutic option. In this work, we isolated and assembled a five-member cocktail of wild phages against Acinetobacter baumannii and demonstrated therapeutic efficacy in a mouse full-thickness dorsal infected wound model. The cocktail lowers the bioburden in the wound, prevents the spread of infection and necrosis to surrounding tissue, and decreases infectio ...[more]

PMID: 27431214

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Project description:Patients recovering from traumatic injuries or surgery often require weeks to months of hospitalization, increasing the risk for wound and surgical site infections caused by ESKAPE pathogens, which include A. baumannii (the ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). As new therapies are being developed to counter A. baumannii infections, animal models are also needed to evaluate potential treatments. Here, we present an excisional, murine wound model in which a diminutive inoculum of a clinically relevant, multidrug-resistant A. baumannii isolate can proliferate, form biofilms, and be effectively treated with antibiotics. The model requires a temporary, cyclophosphamide-induced neutropenia to establish an infection that can persist. A 6-mm-diameter, full-thickness wound was created in the skin overlying the thoracic spine, and after the wound bed was inoculated, it was covered with a dressing for 7 days. Uninoculated control wounds healed within 13 days, whereas infected, placebo-treated wounds remained unclosed beyond 21 days. Treated and untreated wounds were assessed with multiple quantitative and qualitative techniques that included gross pathology, weight loss and recovery, wound closure, bacterial burden, 16S rRNA community profiling, histopathology, peptide nucleic acid-fluorescence in situ hybridization, and scanning electron microscopy assessment of biofilms. The range of differences that we are able to identify with these measures in antibiotic- versus placebo-treated animals provides a clear window within which novel antimicrobial therapies can be assessed. The model can be used to evaluate antimicrobials for their ability to reduce specific pathogen loads in wounded tissues and clear biofilms. Ultimately, the mouse model approach allows for highly powered studies and serves as an initial multifaceted in vivo assessment prior to testing in larger animals.

Project description:Phages have emerged as prime suspects in the adaptation of pathogens to new hosts and the emergence of new pathogens or epidemic clones. Here we describe the genomic features of “swarms” of three related prophages (Ab105-1ϕ, Ab105-2ϕ and Ab105-3ϕ) present in the ST-2 epidemic clone of Acinetobacter baumannii clinical strain Ab105 GEIH-2010 and not present in genetically related Ab155 GEIH-2000 strain isolated 10 years before. The “Quasicore genome” of Ab105-1ϕ, Ab105-2ϕ and Ab105-3ϕ prophages revealed genes that promote bacterial-host fitness. The results of microarray analysis under stress conditions, SOS response and Quorum Sensing (QS) activation revealed 42% and 21% of genes expressed by Ab105-2ϕ and Ab105-3ϕ prophages (which produce bacterial lysis) in the first case and underexpression of these genes from prophages in the second case. Hence, the QS system plays a major role in the evolution of phages in their natural hosts and environments. Interestingly, in host-virus interactions, RT-PCR showed several mechanisms of overexpression of the SOS response in relation to phage defence mechanisms: i) SAM or AdoMet-MTase (methyltransferases) and MazG protein (pyrophosphohydrolase) associated with phage defence in response to bacterial attack; ii) eukaryotic-like protein kinase (glutamate 5-kinase) associated with prevention of secondary infection by the same or a closely related virus. Overexpression of secretory virulence factors such as oxidoreductase (DsbA-like), anfo-nitrogenase and chromosome segregation proteins were also observed. Moreover, under iron-deficient growth, there was an overexpression by RT-PCR of the a new interesting cluster of genes located following a “Moron” organization in the Ab105-3ϕ prophage being associated with iron uptake systems (Xanthine dehydrogenase gene cluster, Anthranilate operon, ABC transporter and TonB dependent receptor). In conclusion, study of the co-evolution of phages (virus) and bacteria may be essential in the search for means of combatting multi-resistant epidemic clones. Two parental clinical strains of A. baumannii (90% identity, indicated by PFGE, and ST2, indicated by Multilocus Sequence Typing, MLST) isolated in the same Intensive Care Unit (ICU) of a Spanish hospital, in 2000 and 2010, during the “I Multicenter Study GEIH-REIPI-Ab-2000” (Ab155 GEIH-2000) and “II Multicenter Study GEIH-REIPI-Ab-2010” (Ab105 GEIH-2010), respectively. Three replicates from RNA of the AB105 GEIH-2010 strain x 2 conditions (SOS response by Mitomycin C) and (Quorum Sensing activation by AHLs mixture).

Project description:Acinetobacter baumannii is a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistant A. baumannii strain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates of A. baumannii from a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combating A. baumannii infections.

Dataset Information

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

Publications

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections.

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