Enhancement of Penicillin-Induced Lysis in <I>Sarcina Lutea</I>

555

Enhancement of Penicillin-Induced Lysis in Sarcina lutea by Lysozyme

JOSEPH M. KORNFELD

Laboratory Division, Connecticut State Department ofHealth.

Hartford, Connecticut06101 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/8/555/1649545/0362-028x-40_8_555.pdf by guest on 26 September 2021

(Received for publication January 21, 1977)

ABSTRACT penicillin are detected when lysozyme is added to the Using either paper disc or cylinder plate methods for detection of detection system. penicillin, addition of egg-white lysozme (N-acetylmuramide glycano hydrolase, E. C. 3.2.1.17) to seeded agar caused production of larger MATERIALS AND METHODS zones of inhibition. The increased zone size enables detection of lesser The procedures used were modified from the established protocols for quantities of the antibiotic. It is suggested that this modification be the assay of penicillin in various materials (2). Antibiotic Agar #4 (BBL) considered for use in detection of low levels of the antibiotic. in 100-ml volumes was seeded with 0.03 ml of a suspension of S. lutea (ATCC 9341) prepared by washing the surface of an overnight slant Under regulations of the United States Food and Drug culture with 5 ml of physiological saline. The seeded agar was agitated to ensure homogeneity and maintained at 49 C for not more than 30 Administration (FDA), presence of penicillin in milk min before use. Freshly prepared lysozyme solution (Egg white, Sigma, constitutes adulteration. Since the current FDA regula 3x crystallized) in phosphate buffer, pH 6.0 was added to 100 x 15 mm tions specify a zero-tolerance for the antibiotic in milk plastic petri plates and the total volume adjusted to 1 ml with and milk products (4), it follows that any milk which additional buffer. In control plates, only buffer was added. The molten, contains any penicillin, no matter how little, is seeded agar was then added to the petri plates in 4-ml volumes and rapidly mixed with the buffer and/or lysozyme. Originally, the pre· adulterated. From a practical point of view, however, it is scribed base layer of 10 ml of Antibiotic Agar #1 was omitted to enable the lowest concentration of the antibiotic which is use of stainless steel cylinders. In the presence of both the base layer and detectable by the prevailing technology which determines cylinders, the petri dish cover could not rest on the lip of the petri dish the adulteration status. base resting instead on the upper edges of the cylinders. Since then, it For detection of penicillin in milk or other foods, the has been shown (5) that omission of the base layer enhances the zone size response to penicillin. After the plates solidified, standards and most widely used procedures are microbiological plate samples were applied to the surface. When cylinders were used, each assays, employing either Sarcina lutea or Bacillus subtilis contained eight drops of the appropriate penicillin solution; one as the indicator organism. The use of B. subtilis enables cylinder always contained only the diluent in which the penicillin more rapid detection, producing readable results in standards were prepared. about 4-5 h. The level of sensitivity obtained with this Penicillin standards were prepared by dissolving a 400,000-unit tablet of Penicillin G (Pfizer) in pH 6.0 buffer and diluting the organism, however, is such that it is difficult to detect suspension to give final concentrations of 0.1, 0.05, 0.03. 0.015, 0.0075. concentrations below 0.03 unit/ml of milk. In contrast, and 0.005 unit/mi. The standards were maintained at 10 C and were the S. lutea method requires overnight incubation, but discarded after 1 week. enables detection of the antibiotic at levels as low as All plates were incubated in an upright position at 32 C, and 0.00125 u/ml. It has been claimed by Wright (8) that the following overnight incubation, zones of inhibition were estimated. S. lutea method was sensitive to 0.005 unit. However, in RESULTS AND DISCUSSION our experience, the method never detects so low a level, and even 0.0125 u/ml is not detected consistently. When lysozyme (0.5 mg) was incorporated into the Nevertheless, the S. lutea · cylinder plate method has seeded growth medium, zones of inhibition produced by been adopted by the FDA as the official assay procedure commercially supplied penicillin discs were dramatically for detection of penicillin in milk '-1). enlarged(Center, Fig. 1). It must be emphasized that in It is likely that no assay procedure will ever reach the the experiment pictured, a single bottle of seeded agar zero tolerance adopted by the FDA. However, modifica was used to produce both plates, that both plates tions of the assay system should enable detection of contained the same volume of medium, and that both lower concentrations than current procedures allow. penicillin discs were taken from a single package. The This paper presents evidence that lesser quantities of zones of inhibition are caused by the penicillin, but 556 KORNFELD

dark background is visible through the agar at all the sites formerly occupied by the cylinders, with the sole exception of the diluent site. On the plate devoid of lysozyme, site transparency ends at 0.0075 u/ml. In the assay of penicillin diluted in milk, these transparent sites would not be observed since deposition of milk components would render them opaque. In addition to opacity, use of milk as diluent also causes a reduction in zone diameters, presumably because of the binding of penicillin to milk proteins with a concommitant reduction in the concentration of the free, diffusible antibiotic. Reduction of zone diameters because of

protein binding has previously been reported for Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/8/555/1649545/0362-028x-40_8_555.pdf by guest on 26 September 2021 cefazolin diluted with a variety of body fluids (6). Notwithstanding any protein binding when milk serves as the diluent for penicillin, the zone sizes in the Figure l. Zones of inhibition produced in the absence (left) and lysozyme plates are always larger than those on the presence (right) of lysozyme. The jour peripheral discs on each plate contained no penicillin. The disc in the center of each plate contained control plate for equivalent penicillin concentrations. 0. 05 unit of penicillin G. In experiments typified by results in Fig. 2, diameters of zones of inhibition were plotted against the log of the enhancement of the size of the zone may only be penicillin concentration used to generate dose-response attributed to the lysozyme incorporated into the agar. curves. Characteristic results are shown in Fig. 3. The When the cylinder procedure for the assay of two curves generally parallel each other, with the antibiotics was substituted for the disc procedure, results lysozyme curve displaced to the right. Since the cylinders shown in Fig. 2 were obtained. Starting at the bottom of themselves impose a limit on zone size, an intercept line each plate and moving clockwise, the concentrations of has been drawn at 8 mm, the diameter of the cylinders. penicillin G used were (in unit/ml) 0.05, 0.00 (diluent),

...... 08 ..J 2 /;· ;· ...... t/) .01 1-z ·' . ::l /lj z I 0 I / 1-c 1/I /' a:: .OJ 1/ I 1-z 11.1 z(.) !;· 0 .008 ~ 0 I Figure 2. Zones of inhibition produced in the absence (left) and 1 presence (right) of lysozyme, using the cylinder plate procedure. The z I concentrations of penicillin used were, starting at the bottom and :::i I moving clockwise (in unitlmn 0.05, 0.()() (diluent), 0.03, 0.015, 0.0075, ..J I (3 I and 0.005. z I 11.1 I 0,03, O.ot5, 0.0075, and 0.005. Once again, both plates Q. I pictured were prepared from a single bottle of seeded I agar. In the plate at the left, which contained no I lysozyme and served as the control, distinct zones of inhibition are found around the sites of the cylinders 0 10 20 30 40 which had contained 0.05,0.03, and O.ot5 unit of penicil ZONE SIZE (mm.) lin/mi. In the plate at the right, which contained 0.5 mg Figure 3. A characteristic dose response curve for the S. lutea-peni· of Jysozmye, zones of inhibition are found at all cillin system showing the effict of lysozyme in enhancing the sensitivity concentrations of penicillin except 0.005 u/ml. It is of the assay. The upper curve represents the control; the lower, solid noteworthy that in the plate containing lysozyme, the line represents results obtained in the presence of lysozyme. LYSIS OF SARCINA LUTEA BY LYSOZYME 557

The intercept of each curve with the 8-mm line indicates enable lysis only at the relatively high penicillin the minimum penicillin concentration detectable by each concentrations which exist within the lytic zone, but not procedure. Although there is variation in the 8-mm at the lesser concentrations which obtain more distal to intercept on a day to day basis, the addition of lysozyme the center of diffusion. When lytic activity is augmented to the agar always enables the detection of 0.0075 u/ml by the exogenous addition of glycosidases, lysozyme in and frequently detects as little as 0.005 u/ml. Using the this instance, additional glycosidic bonds in the cell routine cylinder procedure, in which lysozyme is not walls of the bacteria within this region of low penicillin added, both these concentrations would be missed. concentration are destroyed, weakening the cell walls Use of S. lutea as the indicator organism was and making them unable to withstand the osmotic force particularly fortuitous. Since the use of lysozyme of the cell. Additional lysis then occurs in this area and appeared to render it more sensitive to penicillin, it was the zone size is increased. thought that other organisms of either equivalent or Irrespective of any rationale concerning the mech

lower sensitivity to penicillin might be made more anism of the phenomenon, it is clear that use of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/8/555/1649545/0362-028x-40_8_555.pdf by guest on 26 September 2021 sensitive in the presence of the enzyme. A number of exogenous lysozyme enhanced the sensitivity of S. lutea other organisms were examined, including Micrococcus in the cylinder plate method. Consequently, its use is agilis, Micrococcus denitrificans, Micrococcus roseus, indicated in all systems which are concerned with the Micrococcus lysodeikticus, Micrococcus diversus, Micro detection of extremely low levels of penicillin. coccus rubens, and Bacillus subtilis. Of these, some were insensitive to penicillin with no increased sensitivity in the presence of lysozyme and others were so sensitive to lysozyme that their use was contraindicated. Some REFERENt'ES organisms which were sensitive to penicillin showed no 1. Ghuysen, J. M. M. L. B. Frere, M. N-D. Dusart, J. Coyette, A. added sensitivity in the presence oflysozyme. To date, no Marquet, H. R. Perkins, and M. Nieto. 1975. Bacterial transpepti· organism which has been tested has proven superior to S. dases and penicillin. Bull. lnst. Pasteur 73:101-140. lutea as the indicator organism. 2. Grove, D. C., and W. A. Randall. 1955. Assay methods of antibio· The evidence presented here clearly indicates that tics. Medical Encyclopedia, Inc. pp. 14-16. lysozyme enhances the effect of penicillin on this 3. Kilpatrick. G. W. 1975. Unnumbered FDA memorandum. FDA adoption of the cylinder plate method for detection of penicillin in organism. However, the mechanism of the enhancement milk and milk products. May 28. is obscure and the subject of speculation. Although the 4. Kilpatrick, G. W. 1975. Unnumbered FDA memorandum. Pro specifics of cell death mediated by antibiotics which cedure for detection of penicillin in milk and milk products. Sep interfere in cell wall synthesis are poorly understood, it tember 4. appears that lysis results from the concerted action of 5. Kornfeld,J. M., and J.J. Karolus.1977. The effect of the base layer in the cylinder plate method for analysis of penicillin. J. Food Prot. two separate activities: (a) inhibition of the formation of 40:000-000. peptide bridges between peptidoglycan strands (1), and 6. Peterson, L. R., E. A. Schierl, and W. H. Hall. 1975. Effect of pro (b) hydrolysis of specific bonds in the preexisting murein tein concentration on antibiotic assays. Antimicrob. Agents Che matrix, mediated by autolysins. Among the autolysins mother. 7:540-542. cited as involved in the hydrolysis are various peptidases, 7. Rogers, H. J., and C. W. Forsberg. 1971. Role of autolysins in the killing of bacteria by some bactericidal antibiotics. J. Bacteriol.108: amidases, glucosaminidases, and glycosidases (7). 1235-1243. It is proposed that in the phenomenon reported here, 8. Wright, W. W. 1962. Rapid microbiological disc assay for penicillin the level of endogenous autolysin activity is sufficient to in milk. J. Assoc. Off. Agr. Chern. 45:307-310.