Synergistic Killing of Streptococcus pneumoniae with the Bacteriophage Lytic Enzyme Cpl-1 and Penicillin or Gentamicin Depends on the Level of Penicillin Resistance

Abstract

A combination of Cpl-1, a bacteriophage lytic enzyme, and penicillin, gentamicin, levofloxacin, or azithromycin was tested against Streptococcus pneumoniae strains with various susceptibilities to penicillin. Activities of Cpl-1 and gentamicin were increasingly synergistic with a decreasing penicillin MIC, while Cpl-1 and penicillin showed synergy against an extremely penicillin-resistant strain.Streptococcus pneumoniae is a leading cause of pneumonia, otitis media, meningitis, and sepsis worldwide, and the increase in antibiotic resistance of pneumococci has limited the number of antimicrobial agents that produce reliable treatment results for these infections. Combination therapy by antimicrobials with different mechanisms of action has been used to treat infections for decades with the goal of producing a wider spectrum of action, preventing the emergence of drug-resistant subpopulations, reducing the dose of a single agent, or achieving a synergistic effect.Lytic enzymes are produced by bacteriophages at the end of their replicative cycle and can digest the bacterial cell wall within seconds. Cpl-1 is a muramidase that specifically cleaves the glycosidic bond between N-acetylmuramic acid and Nacetylglucosamine in the cell wall of S. pneumoniae (5). Purified recombinant Cpl-1 has shown extremely rapid killing of S. pneumoniae in vitro and can effectively eradicate these organisms from the nose or the bloodstream of mice (8, 9).We previously reported the synergistic effect of a combination of two phage lytic enzymes with different enzymatic activities, Cpl-1 and Pal, against S. pneumoniae in vitro, and this effect has been confirmed with mouse sepsis models (6, 10). It has not been shown, however, whether lytic phage enzymes in combination with conventional antibiotics could improve their respective efficiencies against pneumococci. In this study we report the in vitro effect of the simultaneous use of Cpl-1 and penicillin, gentamicin, levofloxacin, or azithromycin against four different strains of S. pneumoniae, with MICs of penicillin indicating a range from sensitive to extremely resistant.We produced and purified Cpl-1 from E. coli DH5␣(pJML6) as described elsewhere (8, 10). Cpl-1 was lyophilized and stored at 4°C. Penicillin and gentamicin were purchased from Sigma (St. Louis, Mo.), levofloxacin was purchased from Ortho-McNeil (Raritan, N.J.), and azithromycin was purchased from Pfizer (New York, N.Y.). Stock solutions were made by resuspending the agents in sterile water.For all the pneumococcal strains tested, we first determined MICs of each antibiotic and Cpl-1 by the macrodilution method, using 2 ml of Mueller-Hinton broth (BBL, Becton Dickinson Microbiology Systems, Cockeysville, Md.) supplemented with 5% lysed horse blood (Cleveland Scientific, Inc., Bath, Ohio). The method is described elsewhere in detail (1). The bacterial strains tested and the respective drug MICs are reported in Table 1. Antibiotic and Cpl-1 interactions were assessed by the checkerboard method with 96-well plates, in which Cp...

Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands

“…Combined application of antimicrobial agents not only takes advantage of synergistic effects but also is expected to reduce the chance of resistant strain development, as two simultaneous mutations are likely required to render a cell resistant to both agents (3,28). Furthermore, combinations of antimicrobials have been shown to act synergistically even against strains highly resistant to one of the components (21). In addition to mixtures of two different PG hydrolases as investigated in this work, other classes of antimicrobials, such as antibiotics (21) and bacteriocins (30), have also been reported to exhibit synergy when combined with endolysins, and such combinations may therefore hold promise for treatment of infections such as bovine mastitis.…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…[58][59][60] The most problematic streptococci are the b-hemolytic group A streptococci (GAS) and group B streptococci (GBS), and the a-hemolytic streptococci Streptococcus pneumoniae and Streptococcus viridans.…”

Elucidation of the Molecular Recognition of Bacterial Cell Wall by Modular Pneumococcal Phage Endolysin CPL-1

“…enzybiotics). In addition, Cpl-1 lysin and Pal amidase encoded by phage Dp-1 act in a synergistic manner in a sepsis mouse model (5); this synergy has also been confirmed in in vitro experiments with Cpl-1 and penicillin or gentamicin (6). Very recently, the creation of a new animal model of otitis media has been reported (7).…”

“…Soaking time was 30 min in all cases except for the 2S5P where the time was 18 h; the concentrations for the ligands were 100 mM for the 2S2P and 25 mM for the 2S5P and the (2S5P) 2 . GlcNAc and MurNAc monosaccharides, (GlcNAc) 2 , (GlcNAc) 4 , and (GlcNAc) 6 oligosaccharides, and the PG analogues MurNAc-(L-Ala-D-isoGln) and MurNAc-(L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala) were also tested without success. The x-ray data sets of the wild-type and Cpl-1 E94Q mutant in complex with 2S2P were collected up to 2.3 and 2.0 Å resolution, respectively, using the graphite monochromatic CuK ␣ ( ϭ 1.5418 Å) radiation generated by an Enraf-Nonius rotating anode generator and a MAR345 image plate detector.…”