Novel Phage Lysin Capable of Killing the Multidrug-Resistant Gram-Negative Bacterium Acinetobacter baumannii in a Mouse Bacteremia Model

Abstract

c 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. Members of Acinetobacter are soil bacteria that frequently colonize the human skin without harm (1). However, in environments in which individuals are immunocompromised or suffer from a variety of wounds (e.g., in hospital settings or on battlefields), Acinetobacter baumannii can cause severe life-threatening infections (2-4). Symptoms of A. baumannii infections range from mild skin wounds and urinary tract infections to more severe conditions, including pneumonia, meningitis, and sepsis (5). A. baumannii is now one of the most common causes of hospital-acquired pneumonia (2) and sepsis; while not common (only 1.3% of all sepsis cases), it is associated with mortality rates of up to 58% (6).One of the main threats from A. baumannii is the high rate of resistance to antibiotics commonly used to treat Gram-negative infections. More than 80% of Acinetobacter species are considered to be multidrug resistant (MDR) (i.e., resistant to at least three classes of antibiotics), resulting in infections with poor clinical outcomes, including high rates of morbidity and death, prolonged hospital stays, and substantial health care expenses (3, 7). In addition, several strains of pan-drug-resistant A. baumannii have been isolated, showing resistance to a wide variety of clinically used antibiotics (8). A. baumannii is also capable of surviving treatments with detergents and disinfectants, dehydration, and UV radiation and thus is difficult to eradicate from surfaces in hospital environments (9, 10). The organism not only is intrinsically resistant to many antibiotics (owing to ␤-lactamases, weak membrane permeability, and efficient efflux systems) but also can readily acquire foreign plasmids and is considered to have a high degree of genetic plasticity (11). Outbreaks caused by MDR Acinetobacter have been reported from hospitals worldwide; more recently, they have become a serious problem in military medical facilities (4). One of ...

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates

“…On the other hand, the activity of the CfP1 phage seems to tolerate higher pH values better than the recombinant lysin, which indicates that other lytic accessory proteins might have an important role in phage antibacterial activity. The ability of a Gram-negative-specific phage lysin to kill cells Bfrom without^has been solely attributed to few enzymes (e.g., Enterobacter phage T4, Bacillus phage PlyL, Acinetobacter phage PlyF307, and Pseudmonas phage OBP lysins) (During et al 1999;Morita et al 2001;Walmagh et al 2012;Lood et al 2015). This effect was associated to the positively charged amino acids located at the enzyme's Ctermini, which is able to disrupt or mediate lysin access to the peptidoglycan, causing subsequent bacteriolysis.…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…Intraperitoneal treatment with 1 mg (»40 mg/kg) of PlyF307 within hours after challenge was able to rescue 50% of the infected mice. 23 Compared to rescuing mice from Gram-positive infections with endolysin as described above, which offer as much as 100% protection from death, 50% rescue from a Gram-negative infection is quite low. This suggests the outer membrane is still a formidable barrier for endolysins applied exogenously.…”

“…Although this multi-domain organization is dominant among endolysins from Gram-positive phage, it is not common among endolysins from Gram-negative phage, [20][21][22] which are generally globular and lack CBDs. 23 The number of EADs in the modular endolysins from Gram-positive-infecting phage also varies, with several having two, each encoding a different catalytic activity. 19,24,25 For instance, the staphylococcal phage phi11 endolysin has both an endopeptidase and an amidase.…”

“…Lood et al screened induced prophages to obtain a diversity of 21 Gram-negative background endolysins. 23 The most potent in vitro, PlyF307 was further tested to protect mice from A. baumannii bacteremia. Due to the rapid onset and progression of infection, most untreated animals died within the first day.…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…Bacteriophages use two independent mechanisms to lyse host cells. Whereas some ssDNA and RNA viruses use a peptidoglycan synthesis inhibitor (Young et al, 2000;Zheng et al, 2009;Tanaka & Clemons, 2012), all of the dsDNA viruses follow a lysin-mediated breakdown of peptidoglycan (Young et al, 2000;Young, 2014;Lood et al, 2015). Endolysin is a peptidoglycan-hydrolysing enzyme that carries out enzymic digestion of the cell wall peptidoglycan at the end of the phage infection cycle, which ensures the release of newly packed phage particles.…”