Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme

Abstract

Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C(1) termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of approximately 10(7) group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 10(7) live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease.

Figure 1

Purification of lysin. (A) Silver-stained SDS/PAGE of purified lysin. Molecular mass of Kaleidoscope (Bio-Rad) standards is given in kilodaltons. (B) A bacterial lawn of group A streptococci D471 treated with a buffer control disk (1) or a disk containing 250 units of purified lysin (2), as described in Materials and Methods. A clearing zone indicates antibacterial activity.

Figure 2

Thin-section electron micrograph of Group A streptococci treated for 15 seconds with lysin. The cell wall is weakened (A), allowing the membrane to extrude through the hole (B) created by lysin. ×50,000.

Figure 3

Lysin activity on various streptococci. Representative streptococcal strains were exposed to 250 units of purified lysin, and the OD₆₅₀ was monitored. The activity of lysin for each strain was reported as the initial velocity of lysis, in −OD₆₅₀/min, on the basis of the time it took to decrease the starting OD by half (i.e., from an OD₆₅₀ of 1.0 to 0.5). All assays were performed in triplicate, and the data are expressed as means ± standard deviations.