JOURNAL OF BACTERIOLOGY, May 1968, p. 1956-1960 Copyright ( 1968 American Society for Microbiology Vol. 95, No. 5 Printed in U.S.A. Electron Microscopy of Proteus vulgaris Exposed to Cephalothin' NICHOLAS M. BURDASH,2 MARY A. EHRLICH, HOWARD G. EHRLICH, AND JOSEPH T. PARISI3 Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania 15219 Received for publication 11 March 1968 Exposure of certain gram-negative bacteria to cephalothin and observation by light microscopy revealed the formation of long filaments and large bodies (T. W. Chang and L. Weinstein, J. Bacteriol. 88:1790, 1964). The degree of morphological change was related to the concentration of cephalothin used. This paper describes some abnormal morphological forms of Proteus vulgaris produced upon exposure to cephalothin and studied by electron microscopy. To obtain morphological changes, filter paper discs saturated with 25,ug of cephalothin per ml (Eli Lilly & Co., Indianapolis, Ind.) were placed on Trypticase Soy Agar (BBL) plates inoculated with P. vulgaris. After incubation at 37 C for 15 hr, zones containing abnormal morphological forms were cut from the plates. Fixation was according to the method of E. Kellenberger, A. Ryter, and J. Sechaud (J. Biophys. Biochem. Cytol. 4:671, 1958). Specimens were embedded in Epon 812 (J. H. Luft, J. Biophys. Biochem. Cytol. 9:409, 1961). Sections were cut on a Porter- Blum MT-1 ultramicrotome, mounted on Formvar-coated copper specimen grids, and examined in an RCA-EMU-3G electron microscope. P. vulgaris grew in three distinct concentric zones. The zone immediately surrounding the cephalothin disc was clear and contained no cells. The next zone appeared cloudy and contained large bodies. The third zone contained heavy growth in which long filamentous forms were found. The area surrounding this zone contained normal cells. Electron microscopy of the long filamentous forms revealed no differences except in length between these forms and normal cells. The long forms (Fig. 1) attained lengths of over 20 times that of normal cells, and there was no evidence of a cell wall or cell membrane partitioning the long forms. The large bodies, regarded as spheroplasts, had spherical shapes with diameters of 6 to 12 times that of normal cells (Fig. 2). Several smaller structures within the spheroplast were observed also. These were spherical or ellipsoidal and varied from 0.1 to 0.5,u in diameter. Some of these structures contained densely packed granular material and seemed to form by a budding process from within the spheroplast (Fig. 3). Connections between these were observed frequently (Fig. 4). Other structures in the spheroplast contained little or no electron-dense material and seemed to form in toto within the spheroplast (Fig. 4). The effects of cephalothin on P. vulgaris in the rormation of long filamentous forms and large bodies appear similar to those produced by penicillin (A. Fleming, A. Voureka, J. R. H. Kramer, and W. H. Hughes, J. Gen. Microbiol. 4:257, 1950). In spheroplasts of Escherichia coli produced by penicillin, the antibiotic prevented the incorporation of mucopeptide into the cell wall with a resulting loss of rigidity and impairment in division (R. G. E. Murray, P. Steed, and H. E. Elson, Can. J. Microbiol. 4:547, 1965). In our study, where the concentration of antibiotic was low, the filamentous forms seemed to have an impairment in their division mechanism although they still retained their rigidity. Where the concentration of cephalothin was high, the cells also lost their rigidity and formed spheroplasts. The chemical nature of the smaller structures within the spheroplasts was not determined. However, in view of the similarities in modes of action of penicillin and cephalothin on cell-wall synthesis (P. E. Reynolds, p. 47, in B. A. Newton I Taken in part from a thesis submitted by N. M. and P. E. Reynolds [ed.], Biochemical studies Burdash in partial fulfillment of for the M.S. degree from Duquesne University, Pittsburgh, Pa. antimicrobial drugs, Cambridge Univ. Press, 2Present address: Department of Microbiology, London, 1966), the smaller structures could be Ohio State University, Columbus, Ohio 53210. accumulations of cellular components unable to 8 Present address: Department of Microbiology, be incorporated into the cell wall. P. Fitz-James University of Missouri School of Medicine, Columbia, and R. Hancock (J. Cell Biol. 26:657, 1965) Mo. 65201. observed in Bacillus megaterium grown in the 1956
VOL. 95, 1968 NOTES 1957 u p0 *ill 4 b.... FIG. 1. Longitudinal sectionz of a long filamentous form with an X 51,000. 1. impairment in its division mechanism.
'j,a; C, tt. ;;wt...,4 y U n.- 1958 NOTES J. BACTERIOL. tex_# * t # t - a*t~~~~~~~ 4zt * }> g s8' fz t - e~~~ szo% *d_s.~~ '9; 2 Ij B s J -X,r +,t,[* +,.._w g w FIG. 2. Cross section ofseveral spheroplasts. X 15,000.
:., fi~~~~~~~~~~~~~~~~~~~el els ''': 'X4'''' ':r: S _~~~M. VOL. 95, 1968.NOTES 1959 O. _I..... :~~~~~~~~~~~~~~~4. Avf %g!w t.~~~~~~~~~..._ ; g.., ~~~~~~,R,, X:'w,.!6... i 2t] 'm'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... s... w'1r. ~~~~f,ii,s.a:! SW 1 LW. ts FI.: 3.sBuddin w5ithin peols ofa smler stutr desl pake with graula maera.:x 80 F. 0.12. w 0 i : u lt=.- "..m,.. :., 77.,.f ': i 1, rf Iii W"F. -.: A..A.4, e,4"r.'' S A... ssv..n FIG. 4. Connection between a small structure densely packed with granular material and other small structures whiich have little electron-dense material and are formed in toto within the spheroplast. X 57,000.
1960 NOTES J. BACTERIOL. presence of penicillin the accumulation of mucopeptide, which they concluded was unorganized cell-wall material. In contrast, K. W. Knox, M. Vesk, and E. Work (J. Bacteriol. 92:1206, 1966) observed in a lysine-requiring mutant of E. coli grown in limited lysine the formation of blebs of lipopolysaccharide (LPS) that also resemble the smaller structures in our study. These investigators concluded that this LPS accumulated as a result of the unbalanced growth of the cell wall due to restricted protein synthesis imposed by lysine limitation. Our observations suggest that cephalothin is affecting the synthesis of the cell wall of P. vulgaris and support the finding of similarities in modes of action of penicillin and cephalothin. We thank Eli Lilly & Co. for generously supplying the cephalothin.