Susceptibility of methicillin-resistant Staphylococcus aureus to lysostaphin

M M Huber, T W Huber
1989 Journal of Clinical Microbiology  
were as susceptible to lysis by lysostaphin as methicillin-susceptible S. aureus controls were. The staphylolytic enzyme lysostaphin was discovered by Schindler and Schuhardt (9) and was shown to lyse staphylococci but to be inactive against all other organisms (3, 6, 9) . The basis for the specificity of lysostaphin appears to be its peptidolytic action on pentaglycine cross-linkages, which are peculiar to the cell walls of staphylococci (4). Schleifer and Kloos (10) proposed the use of
more » ... d the use of lysostaphin susceptibility for differentiation of staphylococci from micrococci. Baker (1) found that most Staphylococcus aureus isolates tested were lysed by lysostaphin but that lysis of coagulase-negative staphylococci was inconsistent. Zygmunt et al. (15) found that over 400 strains of multiply resistant methicillinsusceptible S. aureus (MSSA) were lysèd by lysostaphin. Conversely, cross-resistance to seven antibiotics did not arise in laboratory-selected lysostaphin-resistant mutants. MSSA seems to be inherently susceptible to lysostaphin, but the literature is inconclusive on lysostaphin susceptibility of methicillin-resistant S. aureus (MRSA). Sabath et al. (8) reported diminished lysis but Seligman (11) reported enhanced lysis of MRSA by lysostaphin. Zygmunt et al. (14) reported that five strains initially isolated as MRSA were susceptible to lysostaphin but, on repeat testing, were found to have lost their methicillin resistance. No more than a dozen isolates were tested in any study, so no systematic study of the susceptibility of MRSA to lysostaphin has been reported. We report testing a large number of clinical isolates of MRSA collected over a 4-year period to ascertain whether methicillin resistance affects the credibility of lysostaphin as a taxonomic criterion. A total of 111 clinical isolates of MRSA were collected from March 1983 to April 1987. The 111 isolates were single isolates from 99 patients and 2 isolates each from 6 patients. MRSA isolates from the same patient were different as follows: 3 sets were collected from different sites; 1 set was from the same site but a month apart; 1 set had different antibiograms; 1 set had no difference. Antimicrobial susceptibility patterns were available for 94 of the 111 isolates. The organisms segregated into 15 resistance patterns, with 33% falling into the most common antibiogram type, 30% falling into another type, and 37% scattered among the other 13 antibiogram types. While some testing of the same strain probably occurred, the diversity of antibiograms indicates that the 111 MRSA isolates represent more than a single endemic strain persisting for 4 years in our hospital. Methi-* Corresponding author. cillin resistance was determined by the disk diffusion procedure of Boyce (2) and corroborated by AutoMicrobic System Gram-Positive Susceptibility MIC (Vitek Systems, Inc., Hazelwood, Mo.) results. Methicillin resistance was reconfirmed for 12 random isolates (oxacillin MIC, >8 ,ug/ml) by Sceptor Gram-Positive MIC panels (Johnston Laboratories, Inc., Towson, Md.). Organisms were maintained at -70°C in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) plus 20% glycerol in 2-ml vials. Vials were thawed, and organisms were streaked to 5% sheep blood agar plates (BBL). S. aureus ATCC 25923 was the methicillin-susceptible (MSSA) control organism. Lyophilized lysostaphin (Mead Johnson, Evansville, Ind.) was diluted with sterile distilled water to a concentration of 1,000 1ig/ml. Rehydrated lysostaphin was stored at -20°C until use. The 24-h growth from sheep blood agar plates was suspended to produce a heavy suspension in 5.5 ml of phosphate-buffered saline (pH 7.3). Suspended organisms (2.5 ml) were dispensed into two tubes (12 by 75 mm). Lysostaphin (25 Fil) was pipetted into test suspensions, and 25 ,ul of phosphatebuffered saline was added to the control tubes. The final concentration of lysostaphin was 10 ,ug/ml. Optical density (OD) of test and control suspensions was determined by using a Coleman Junior spectrophotometer (The Perkin-Elmer Corp., Norwalk, Conn.) at 520 nm. OD was determined after 0, 10, 20, 30, 40, and 60 min of incubation in a 37°C water bath. The average percent reduction in turbidity for 111 isolates of MRSA is represented by the closed-square curve in Fig. 1 . No appreciable autolysis was shown by MRSA isolates or MSSA control suspensions. Comparison of curves indicates that every MRSA isolate was lysed by lysostaphin, and the average rate of lysis indicated MRSA isolates (closed squares) to be as sensitive to lysis as were MSSA controls (open circles). The shaded area shows the range of reduction in turbidity seen in lysis tests. The width of the range oflysis curve could be due to variation in strain susceptibility or the turbidity of initial suspensions. The effect of initial turbidity on the rate of lysis of MRSA by 10 p.g of lysostaphin per ml is shown in Fig. 2 . Heavy suspensions showed slower clearing, as would be expected with enzyme saturation. Heavy suspensions did continue to clear with time. The heaviest suspension showed the slowest rate but reached 93% reduction of turbidity in 90 min. Initial turbidities ranged from 0.361 to 1.067 OD units. Optimal turbidity appeared to be greater than 0.4 but less than 0.9 OD units and could be achieved by using either McFarland turbidity 1122 on September 12, 2018 by guest
doi:10.1128/jcm.27.5.1122-1124.1989 fatcat:juyproflf5c73eghtjyvq3p7um