Author Institution: Division of Infectious Diseases, Department of Medicine, The Ohio State University College of Medicin

Perkins, Robert L.

Prior, Richard B.

Seed, Thomas M.

The Ohio Journal of Science

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BRIEF NOTESURFACE CHARGE DETERMINATION OF PROTEUS MIRABILISEXPOSED TO CARBENICILLIN1RICHARD B. PRIOR, THOMAS M. SEED* and ROBERT L. PERKINS, Division of InfectiousDiseases, Department of Medicine, The Ohio State University College of Medicine, Columbus,OH 43210OHIO J. SCI. 77(5): 210, 1977Proteus mirabilis undergoes a series ofpredictable morphological changes whenexposed to different concentrations ofcarbenicillin (Perkins and Miller 1973).At low drug concentrations septum for-mation is inhibited but cell growth is notaffected; thus, filamentation occurs. Athigher drug concentrations, both septumformation and cell growth are inhibitedand spheroplast formation occurs. Inour experiments, the surface charge dis-tribution of P. mirabilis exposed to vari-0.5/jfmFIGURE 1. Transmission electron micrograph of control P. mirabilis exposed to vary-ing concentrations of the minimal inhibitor concentration of carbenicillin stainedwith a positvely charged collodial-iron mixture showing attachment of iron particles(arrows) to surface of cell walls (C\V).Manuscript received March 4, 1977 and inrevised form as a note April 28, 1977 (#77-27).2Part of this work was done by Thomas M.Seed at the Blood Research Laboratory,American National Red Cross, Bethesda, MD20014. His present address is: Division ofBiological and Medical Research, Argonne Na-tional Laboratory, Argonne, IL.ous concentrations of disodium carbeni-cillin was studied using a positivelycharged colloidal-iron stain and transmis-sion electron microscopy.P. mirabilis (minimal inhibitory con-centration carbenicillin, 0.49 /xg/ml) wascultured in trypticase-soy broth (TSB)210Ohio J. Sci. SURFACE CHARGE OF PROTEUS MIRABILOS 211for 4 hr and then 1 ml aliquots weretransferred to 9 ml TSB containing vari-ous concentrations of carbenicillin. Finalcarbenicillin concentrations equivalent to0.1, 1.0, 10, and 100 times the minimalinhibitory concentration (MIC) werestudied. The antibiotic-containing cul-tures were incubated at 37°C for 4 hr.Control cultures of P. mirabilis exposedto drug-free diluent were treated in anindentical manner. Following exposureto the various drug concentrations, thecells were fixed in 1% glutaraldehyde in0.05M phosphate buffer pH 7.3 with 4%sucrose as previously described by Priorand Perkins (1974). The cells were thenwashed and exposed to an acetic acid-iron colloid mixture at pH 1.8 for 15 minat 25°C. The colloidal iron-staining re-agents were prepared and utilized ac-cording to the methods of Gasic et al(1968). Then the cells were washed firstin acetic acid, then in distilled water, andwere post-fixed subsequently in 1%osmium tetroxide. Following fixation,the cells were prepared for electron micro-scopy by the methods previously reportedby Seed et al (1974) and ultrathin sec-tions were examined with a Zeiss EM 9Selectron microscope.FIGURE 2. Transmission electron micrographs of P. mirabilis exposed to various concentrations ofdisodium carbenicillin and subsequently stained with a positively charged collodial-iron mixture.(A) Exposure to 0.1 times the MIC showing cytoplasmic vacuoles and attachment of iron particles(arrow) to surface of cell wall (CW). (B) Exposure to 1 times the MIC showing cytoplasmicvacuoles and attachment of iron particles to filamentous form (arrows). (C) Exposure to 10 timesthe MIC showing iron particle aggregates within cytoplasm and cytoplasmic vacuoles (arrows) aswell as attachment to cell wall surface. (D) Exposure to 100 times the MIC showing filamentousform with iron particles attached to cell wall surface and within cytoplasm (arrows). A largespheroplast is also shown which lacks a cell wall and also attachment of iron particles to cyto-plasmic membrane. (MIC = minimal inhibitory concentration).212 RICHARD B. PRIOR ET AL Vol. 77The control P. mirabilis (fig. 1) hadrelatively uniform distribution of ironparticles (arrows) attached to their cellwall surface (CW). oThese iron particlesmeasured about 40A in diameter. At0.1 times the MIC of carbenicillin thecells had surface charge distributionsimilar to that of the control cells (fig.2A). Small cytoplasmic vacuoles seenin these cells were attributed to the effectsof the drug because previously publishedreports have described similar vacuoles(Fitz-James and Hancock 1965). At 1times the MIC of carbenicillin the typicalfilamentous forms were observed (fig.2B) and the surface charge distributionwas similar to that observed in cellstreated with the lower drug concentra-tion. Cytoplasmic vacuoles along theinner portion of the cell wall were alsopresent. At exposure to 10 times theMIC of carbenicillin, filamentous formswere also observed and the attachmentof iron particles to a typical filamentousform is shown in figure 2C. Aggregatesof iron particles were observed within thecytoplasm and within several vacuoles ofcells treated with this higher drug con-centration (fig. 2C). Penetration of theiron particles into the cytoplasm may becaused by change in permeability of thecell wall due to the action of the drug. Afilamentous form and spheroplast pro-duced during exposure to 100 times theMIC of carbenicillin are shown in figure2D. Iron particles became attached tothe cell surface and also aggregatedwithin the cytoplasm of the filamentousform. The failure of iron particles toattach to the large spheroplast formed at100 times the MIC of carbenicillin (fig.2D) indicates lack of a negative chargeon the cytoplasmic membrane.The present study demonstrated littlealteration in cell-wall charge due to car-benicillin, greater penetration of ironparticles at high drug concentrations,and the absence of iron particle attach-ment with loss of cell wall. Studies toevaluate the positive charge distributionusing negatively charged iron colloidsand to determine the chemical nature ofthe charges by selective enzymatic treat-ment of the cells are in progress. Webelieve this cytochemical approach maycontribute to a better understanding ofthe mode of action of cell-wall-activeantibiotics.LITERATURE CITEDFitz-James, P. and R. Hancock 1965 Theinitial structural lesion of penicillin action inBacillus megaterium. J. Cell Biol. 26: 657-667.Gasic, G. T., L. Berwick and M. Sorrentino1968 Positive and negative colloidal iron ascell surface electron stains. Lab. Invest. 18:63-71.Perkins, R. L. and M. A. Miller 1973 Scan-ning electron microscopy of morphologicalalterations in Proteus mirabilis induced bycephalosporins and semisynthetic penicillins.Can. J. Microbiol. 19: 251-255.Prior, R. B. and R. L. Perkins 1974 Artifactsinduced by preparation for scanning electronmicroscopy in Proteus mirabilis exposed tocarbenicillin. Can. J. Microbiol. 20: 794-795.Seed, T. M., M. Aikawa, C. Sterling and J.Rabbege 1974 Surface properties of extra-cellular malaria parasites: morphological andcytochemical study. Infect. Immun. 9: 750-761.

Brief Note  Surface Charge Determination of Proteus Mirabilis Exposed to Carbenicillin

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Brief Note Surface Charge Determination of Proteus Mirabilis Exposed to Carbenicillin

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