Novel phage lysin capable of killing the multidrug-resistant gram-negative bacterium Acinetobacter baumannii in a mouse bacteremia model

Abstract

Acinetobacter baumannii, a Gram-negative multidrug-resistant (MDR) bacterium, is now recognized as one of the more common nosocomial pathogens. Because most clinical isolates are found to be multidrug resistant, alternative therapies need to be developed to control this pathogen. We constructed a bacteriophage genomic library based on prophages induced from 13 A. baumannii strains and screened it for genes encoding bacteriolytic activity. Using this approach, we identified 21 distinct lysins with different activities and sequence diversity that were capable of killing A. baumannii. The lysin (PlyF307) displaying the greatest activity was further characterized and was shown to efficiently kill (>5-log-unit decrease) all tested A. baumannii clinical isolates. Treatment with PlyF307 was able to significantly reduce planktonic and biofilm A. baumannii both in vitro and in vivo. Finally, PlyF307 rescued mice from lethal A. baumannii bacteremia and as such represents the first highly active therapeutic lysin specific for Gram-negative organisms in an array of native lysins found in Acinetobacter phage.

FIG 1

Examples of inducible phages from A. baumannii and generation of a phage lysin library. (A) Inducible bacteriophages from A. baumannii clinical isolates were negatively stained with 2% uranyl acetate and visualized by electron microscopy. They are typical of siphoviruses (strains 1790 and 1796) or myoviruses (strain 1794). Bars, 100 nm. (B) DNA was extracted from the phages, and a phage genomic library was established. Clones were screened for their ability to generate clearing zones on soft agar plates containing A. baumannii (arrows).

FIG 2

Identified lysins and their activity versus A. baumannii. (A) All constructs with activity against A. baumannii (PlyF301 to PlyF376) were sequenced, and a phylogenetic tree was generated using the software MacVector (unweighted pair group method with arithmetic mean [UPGMA], with Poisson correction). Three main classes of proteins were identified based on domain organization, including proteins with (i) a TIGR02594 domain, (ii) a catalytic domain and a binding domain, and (iii) a lysozyme domain. (B) The 21 different constructs were screened for activity versus 13 different A. baumannii clinical isolates. Crude lysates (10 μl) were added to a soft agar plate with A. baumannii and incubated for 1.5 h at room temperature each day, while being kept at 4°C the rest of the time. Plates were incubated until bacterial growth and clearing zones were visible (4 to 5 days). Clearing zones larger than the original spot of crude lysate were scored. Numbers above the bars, numbers of strains for which the specific lysin was most efficient.

FIG 3

Purification and activity of A. baumannii phage lysin PlyF307. (A) A. baumannii phage lysin PlyF307 (arrow) was purified by a combination of ion-exchange chromatography and size exclusion chromatography, which resulted in purity of ∼95%, as observed visually. (B) PlyF307 is composed of a single lysozyme domain, involving amino acids 3 to 141. The C-terminal part of the lysin has a high positive net charge (charge of +7), while the N-terminal part is closer to a neutral net charge (charge of +1). (C) The activity of PlyF307 was investigated by determining its effects against both exponentially growing cells (Log) and stationary-phase cells (Stat), by incubating the samples for 2 h at 37°C with 100 μg/ml PlyF307. Black bars, samples without the addition of PlyF307; gray bars, samples with the addition of PlyF307.

FIG 4

(A and B) Optimal conditions, i.e., pH optimum (A) and NaCl optimum (B), for PlyF307 determined using A. baumannii isolate 1791. (C) Killing activity of PlyF307 against 13 A. baumannii clinical isolates. All experiments were conducted using exponentially growing bacteria that had been washed and resuspended to ∼10⁶ CFU/ml in sodium phosphate buffer (pH 6.0). PlyF307 (100 μg/ml) was added and the samples were incubated for 2 h before serial dilutions were plated for CFU counting. Black bars, samples without the addition of PlyF307; gray bars, samples with the addition of PlyF307. Horizontal lines, limits of detection in the different experiments. All experiments were conducted in triplicate, and results are presented as mean ± SD.

FIG 5

Ability of PlyF307 to degrade A. baumannii biofilms in vitro and in vivo and to rescue mice from lethal bacteremia. (A) A. baumannii biofilms were formed in vitro on catheters for 24 h before being treated in vitro with PlyF307 for 2 h. For the in vivo samples, whole catheter pieces with 2-day-old biofilms were implanted subcutaneously in the backs of mice. After 24 h, two doses of 1 mg of PlyF307 or buffer were administered subcutaneously, 4 h apart, at the implanted site. Two hours after the last dose, the catheter was removed and sonicated, and the dislodged A. baumannii organisms were plated for CFU enumeration. Black bars, controls; light gray bars, samples treated with PlyF307. (B) A 3-day-old A. baumannii biofilm was established on a catheter and treated for 30 min with 250 μg PlyF307 before being analyzed using scanning electron microscopy. Magnification, ×20,000. (C) Mice were infected i.p. with 10⁸ CFU of A. baumannii. They received a single dose of PlyF307 (1 mg) or buffer i.p. 2 hours later, and they were monitored for survival for 14 days.