Alteration in Bacterial Morphology by Optochin and Quinine Hydrochlorides1

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1 JOURNAL OF BACTERIOLOGY, Jan. 1969, p American Society for Microbiology Vol. 97, No. I Printed in U.S.A. Alteration in Bacterial Morphology by Optochin and Quinine Hydrochlorides1 ARNOLD S. KREGER,2 D. C. SWARTZENDRUBER,3 AND R. H. OLSEN Department of Microbiology, University of Michigan, Ann Arbor, Michigan Received for publication 16 October 1968 Incubation of washed bacterial and ribosomal suspensions with optochin or quinine hydrochloride caused an increase in the turbidity of the suspensions and the appearance of electron-dense cytoplasmic aggregates in the treated cells. These effects were more pronounced with optochin hydrochloride than with quinine hydrochloride, and they did not correlate with the relative sensitivities of different bacteria to growth inhibition by optochin or quinine. Suspensions of washed pneumococci incubated for a short time with high concentrations (1 mg/ ml) of optochin hydrochloride or quinine hydrochloride increase in turbidity (unpublished data). Cellular morphology and chain length, as determined in wet-mount or Gram-strain preparations, were not altered by these treatments. Therefore, to determine whether changes in ultrastructural morphology could be correlated with the increase in turbidity, treated and untreated cells were examined in the electron microscope. Experiments were also done to determine whether the increase in turbidity was specific for the pneumococcus or whether there was a correlation between the phenomenon and the relative sensitivities of different bacteria to growth inhibition by optochin or quinine. This report shows that the increase in turbidity of bacterial suspensions incubated with optochin or quinine hydrochloride is a nonspecific effect of high concentrations of drug, not related to optochin or quinine sensitivity, and is associated with the appearance of electron-dense aggregates in the cytoplasms of treated cells. MATERIALS AND METHODS Organisms. Pseudomonas aeruginosa (1C strain) was obtained from B. W. Holloway (Department of Bacteriology, University of Melbourne, Victoria, Australia) and was maintained on tryptone glucoseyeast extract (TGE) agar containing 0.5% Tryptone (Difco), 0.25% Yeast Extract (Difco), 0.1% glucose, and 1.5% Agar (Difco). This organism was grown in I Taken from a thesis submitted by Arnold S. Kreger to the University of Michigan Graduate School in partial fulfillment of the requirements for the Ph.D. degree. 2 Present address: Department of Microbiology, New York University School of Medicine, New York, N.Y Present address: Oak Ridge Associated Universities, Oak Ridge, Tenn TGE broth or Brain Heart Infusion (BHI, Difco) a 37 C. The isolation of the optochin-resistant pneumococci has been reported (2). The organisms were grown in BHI at 37 C. All other organisms were obtained from the culture collection of the Department of Microbiology, University of Michigan, Ann Arbor. They were grown at 37 C in BHI, with the exception of the Micrococcus lysodeikticus and Lactobacillus acidophilus strains which were grown in Trypticase Soy Broth (TSB, BBL) at 37 C. Determination of minimal growth-inhibitory concentrations of optochin hydrochloride and quinine hydrochloride. Minimal growth-inhibitory concentrations of optochin and quinine hydrochlorides were determined for M. lysodeikticus and L. acidophilus in TSB and for all other organisms in BHI. Fivemilliliter volumes of broth, containing various concentrations of optochin hydrochloride (K & K Laboratories, Plainview, N.Y;) or quinine hydrochloride (Mallinckrodt Chemical Works, St. Louis, Mo.), were inoculated with approximately 106 late logphase cells. The presence or absence of growth was noted after incubation for 2 days at 37 C. Extraction of ribosomes. All procedures were carried out at approximately 5 C. The buffer used was 0.01 M tris(hydroxymethyl)aminomethane hydrochloride (ph 7.6) with 0.02 M magnesium acetate. Cells were harvested from 2 liters of a late log-phase TGE culture of Pseudomonas aeruginosa 1 C. These cells were washed twice and suspended in 20 ml of buffer. The suspension was subjected to sonic disruption for 30 min at full power in a tap water-cooled Raytheon 10-kc magnetostriction oscillator, and the sonictreated material was centrifuged twice at 10,000 X g for 30 min. The supernatant fluid was centrifuged twice at 34,000 X g for 30 min, each time discarding the pellet, and then centrifuged once at 105,000 X g for 2 hr. The final pellet was washed twice, suspended in 20 ml of buffer, dialyzed overnight against 3 liters of buffer, and centrifuged at 10,000 X g for 10 min. The supernatant fluid was decanted and stored at 5 C. 362

2 VOL. 97, 1969 ALTERATION IN BACTERIAL MORPHOLOGY BY OPTOCHIN 363 Electron microscopy. Bacterial pellets were fixed for 15 min in 2.5% glutaraldehyde in 0.15 M phosphate buffer (ph 7.2), washed thoroughly with buffer, post-fixed for 2 hr in 1% Palade's fixative (5), dehydrated in ethyl alcohol, and embedded in Epon 812 (3). Thin sections were stained with aqueous uranyl acetate and lead citrate and were examined with a Philips EM 200 electron microscope. Ribosome suspensions were pipetted onto Formvarcoated grids which were stabilized lightly with carbon. Excess fluid was removed by blotting the grids onto filter paper. Preparations were allowed to air dry prior to examination. Treatment of washed bacteria. Early stationary phase cells were washed twice with 0.05 M phosphate buffer (ph 7.6) and suspended in buffer to an absorbancy at 650 nm of approximately 0.2 (approximately 109 cells per ml). A 0.5-ml portion of glassdistilled water or of the appropriate optochin hydrochloride or quinine hydrochloride stock solution was added to 4.5-ml samples of suspensions, and the mixtures were incubated at 37 C for 15 to 120 min. After measurement of the absorbancy of the suspensions at 650 nm in a Bausch and Lomb Spectronic-20 colorimeter, the cells were sedimented by centrifugation in the cold (5 C) and washed twice with chilled buffer. The pellets were then fixed and examined as described. Treatment of washed ribosomes. Samples (4.5 ml) of a washed ribosome suspension, with an absorbancy of aproximately 0.2 at 650 nm, were incubated for 30 min at 37 C with 0.5 ml of glass-distilled water or of the appropriate optochin hydrochloride or quinine hydrochloride stock solution. After measurement of the absorbancy of the suspensions at 650 nm, small amounts of suspensions were applied to grids and examined by electron microscopy. RESULTS Washed cell studies. Although there was some variation in the extent of the increases in absorbance observed among the different bacteria, no distinct correlation was seen between the sensitivity to growth inhibition of a bacterium and the ability of the drugs to affect the absorbance of the bacterial suspension (Table 1). The effect was greater with optochin hydrochloride than with quinine hydrochloride. Maximal increases were observed by 30 min postincubation, and no decreases were observed over periods up to 120 min postincubation. Untreated pneumococci had a relatively homogeneous dispersion of ribosomes and other granular material in their cytoplasms (Fig. 1); however, the incubation of pneumococci with 1 mg of optochin hydrochloride or quinine hydrochloride per ml resulted in the appearance of electron-dense aggregates in their cytoplasms (Fig. 3). The approximate diameter of these aggregates in cells incubated with optochin hydrochloride was 35 nm and, in cells incubated with quinine hydrochloride, approximately 20 nm. Smaller TABLE 1. Increase in absorbance and growth inhibition of various bacteria by optochin and quinine hydrochlorides Organism Per cent increase (MIC)a Optochin hydrochloride Quinine hydrochloride Optochin-sensitive pneumococcus (0.5) 40 (3) Optochin-resistant 70 pneumococcus.. 80 (70) 45 (300) Optochin-resistant 160 pneumococcus. 90 (160) 35 (600) Staphylococcus aureus (P209) (400) 48 (700) Staphylococcus albus. 71 (400) 55 (700) Streptococcus mitis (70) 69 (300) Micrococcus lysodeikticus (200) 30 (400) Lactobacillus acidophilus (80) 25 (200) Bacillus subtilis (400) 20 (600) Pseudomonas aeruginosa (lc strain) (1,000) 40 (>1,000) Escherichia coli (Snyder strain) (400) 25 (600) Salmonella typhosa (Rawlings strain). 60 (300) 30 (300) Shigella flexneri (300) 0 (300) Aerobacter aerogenes. 80 (500) 25 (600) Serratia marcescens.. 50 (1,000) 20 (> 1,000) Bacterium anitratum.. 60 (60) 20 (200) a Per cent increase in absorbance at 650 nm after incubation at 37 C for 30 min with 1 mg of drug/ml [minimal growth-inhibitory concentration (MIC) in,ug/ml]. aggregates were observed in cells exposed to 500,ug of optochin hydrochloride per ml (Fig. 2). Particles with the appearance of ribosomes were seen in the cytoplasmic aggregates. Similar morphological changes were observed with P. aeruginosa cells. Washed ribosome studies. The absorbance of ribosomal suspensions incubated with 1 mg and 500,ug of optochin hydrochloride per ml increased 59 and 27%, respectively. Incubation with 1 mg and 500 Mg of quinine hydrochloride per ml increased the absorbance by 38 and 16%, respectively. No ribosomal aggregates were seen; however, electron microscopy showed the optochin-treated ribosomes to be retained on the grid more heavily than untreated ribosomes. DISCUSSION O'Brien et al. have reported that quinine binds in vitro to deoxyribonucleic acid bases through

3 FIG. 1. Untreated control optochin-resistant 70 pneumococcus. Note the relatively homogeneous dispersion of ribosomes and other granular material. Line marker denotes distance of 50 nm. X 200,000. FIG. 2. Optochin-resistant 70 pneumococcus incubated at 37 C for 30 min with 500 lag of optochin hydrochloride per ml. Note the presence of small electron-dense cytoplasmic aggregates. The amorphous "patch" in the left corner of the cell has also been observed in untreated control pneumococci. Line marker denotes distance of50 nm. X 180,

4 VOL. 97, 1969 ALTERATION IN BACTERIAL MORPHOLOGY BY OPTOCHIN 365 FIG. 3. Opt-r70 pneumococcus incubated at 37 C for 30 min with I mg of optochin HCI per ml. X 200,000. Note the presence of electron-dense cytoplasmic aggregates, approximately 35 nm in diameter, containing ribosome-size particles. Line marker denotes distance ofso nm. the formation of hydrogen bonds (4). The in vivo formation of ribosomal aggregates and the increased adhesion of in vitro ribosomes after incubation of cells and free ribosomes, respectively, with quinine and its analogue optochin, might then be a macroscopic manifestation of the formation of interconnecting bonds between ribosomal ribonucleic acid bases.

5 366 KREGER, SWARTZENDRUBER, AND OLSEN J. BACTERIOL. Although the concentrations of optochin hydrochloride which are required to produce the increase in absorbance and the appearance of the electron-dense cytoplasmic aggregates are also growth-inhibitory, it seems unlikely that the phenomena are indicative of the primary antipneumococcal action of optochin. If they were, there should have been a correlation between the sensitivity of the different bacteria to growth inhibition by optochin and the amount of drug required to elicit the phenomena in the different bacteria. It is possible, however, that the phenomena are indicative of a secondary mechanism of action of optochin for the pneumococcus or of a primary mechanism for growth inhibition of the more optochin-resistant bacteria. Brzin (1) reported that optochin hydrochloride caused swelling and elongation of growing Bacterium anitratum. No alteration in morphology was observed with other gram-negative bacteria, such as Escherichia coli, Aerobacter aerogenes, Proteus sp., Salmonella typhi, Klebsiella pneumoniae, Serratia marcescens, Pseudomonas pyocyanea, and Pasteurella pseudotuberculosis. We observed that B. anitratum was the most sensitive of all the gram-negative bacteria tested to growth inhibition by optochin and quinine hydrochlorides. ACKNOWLEDGMENT We thank John Freer for helpful suggestions during preparation of the manuscript. LITERATURE CITED 1. Brzin, B Induction of morphological changes in bacteria by optochin. Experientia 21: Kreger, A. S., R. H. Olsen, and M. B. Talmadge Altered diaphorase activity in optochin-resistant pneumococci. J. Bacteriol. 96: Luft, J. H Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: O'Brien, R. L., J. G. Olenick, and F. E. Hahn Reactions of quinine, chloroquine, and quinacrine with DNA and their effects on the DNA and RNA polymerase reactions. Proc. Natl. Acad. Sci. U.S. 55: Palade, G. E A study of fixation for electron microscopy. J. Exptl. Med. 95: Downloaded from on March 19, 2019 by guest