STUART L. HAZELLt* AND DAVID Y. GRAHAM. Digestive Disease Section JJJD, Department ofMedicine, Veterans Affairs Medical Center, and. Baylor College ...
Vol. 28, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1990, p. 1060-1061
0095-1137/90/051060-02$02.00/0 Copyright © 1990, American Society for Microbiology
Unsaturated Fatty Acids and Viability of Helicobacter
(Campylobacter) pylori STUART L. HAZELLt* AND DAVID Y. GRAHAM Digestive Disease Section JJJD, Department of Medicine, Veterans Affairs Medical Center, and Baylor College of Medicine, Houston, Texas 77030 Received 13 November 1989/Accepted 5 February 1990
Helicobacter (Campylobacter) pylori was found to be sensitive to the toxic effects of an unsaturated fatty acid (arachidonic acid). Data are presented that support the hypothesis that exogenous catalase added to basal media enhances the growth of H. pylori by preventing the formation of toxic peroxidation products from long-chain unsaturated fatty acids. We recently demonstrated (3) that the addition of bovine serum albumin (BSA) and catalase to a basal medium was sufficient to both support and maintain a population of Helicobacter (Campylobacter) pylori (2, 6) that had been originally isolated on blood agar. It was proposed that the mechanism by which BSA and catalase promoted the growth of H. pylori was by reduction of the toxic effects of fatty acids, BSA acting by adsorption to short-chain fatty acids and catalase by preventing the formation of toxic peroxidation products from long-chain unsaturated fatty acids (3). An interesting finding of the above-mentioned work was the discovery of a subpopulation of cells that would grow on basal media supplemented only with BSA. This observation suggested that such "catalase-independent" organisms were naturally resistant to toxic peroxidated long-chain unsaturated fatty acids. We therefore decided to test whether long-chain polyunsaturated fatty acids are toxic for H. pylori and whether catalase independence is a marker for increased resistance to such toxicity. Primary isolates of H. pylori maintained on blood agar ("catalase dependent") and a catalase-independent line derived from the same cells were examined for their sensitivity to arachidonic acid. An examination of the protein profiles of these two cell lines was also undertaken. Four fresh clinical isolates stored at -70°C were used for this study. Cells were recovered from the frozen stocks and cultured on 5 to 7% (vol/vol) horse blood agar at 37°C in an environment of 10% C02 in air and 99% relative humidity. Cells were subcultured to Iso-Sensitest agar (Oxoid, Columbia, Mass.) containing BSA (3) or horse blood and passaged every 2 to 3 days (Fig. 1). Sensitivity to arachidonic acid was determined by the method of Knapp and Melly (5). Briefly, cells were washed and suspended in 0.01 M sodium phosphate-buffered saline (pH 7.2) to give a slightly turbid suspension (-106 to 107 CFU/ml). To 10 ml of suspension was added either 3 or 30 ,ul of a stock solution of arachidonic acid dissolved in absolute ethanol (to give a final concentration of 0.01 or 0.1 mM, respectively) or, as a control, absolute ethanol alone. The suspensions were kept at room temperature for 1 h. Viability was determined by the method of Miles and Misra (7),
whereby serial dilutions of the bacterial suspensions were prepared in duplicate, 20-pil drops of each dilution were applied to segments of blood agar plates, and colony counts were performed following incubation. Assays were run in duplicate, and the data from each set were combined for the determination of the mean count and standard deviation. In addition to testing the sensitivity of H. pylori to arachidonic acid, we fractionated cells by the methods of Inouye and Guthrie (4) and Filip et al. (1) for examination of their protein profiles. Bacteria were washed in 0.1 M potassium phosphate buffer (pH 7.35) and disrupted by three cycles through a French pressure cell (Aminco, Urbana, Ill.). Large fragments were removed by centrifugation (17,000 x g for 8 min), and the supernatant was used to obtain soluble proteins and two membrane fractions. The latter were prepared from material solubilized in 5 mM EDTA-0.25% (wt/vol) N-lauroylsarcosine and from the insoluble residue. All three fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in Trisglycine buffer (Eastman Kodak Co., Rochester, N.Y.). Protein bands were stained with Coomassie brilliant blue R. No viable H. pylori cells were found after incubation of either the catalase-dependent or the catalase-independent lines with 0.1 mM arachidonic acid. Following 1 h of incubation with 0.01 mM arachidonic acid, there was a 3- to 4-log decrease in the viability of the catalase-dependent line (as compared with controls). These results contrast with those obtained for the catalase-independent line, which was significantly more resistant to the same concentration of arachidonic acid, with approximately a 1-log decrease in viability over the same period (Fig. 2). These results confirm that the ability of H. pylori to grow on basal medium containing BSA in the absence of whole blood or exogenous catalase (catalase independence) correlates with a reduced sensitivity to arachidonic acid. Catalase independence was able to be modulated. For example, reculturing catalase-independent strains from IsoSensitest agar containing BSA back onto blood agar led to a "loss" of catalase independence in two strains and a reduction in the resistance to arachidonic acid toxicity in two others (Fig. 2). This result and the results of our previous study (3) suggest that catalase independence can be switched on and off or that a proportion of catalase-dependent cells are maintained in the presence of catalase-independent cells. If the latter is correct, then the catalase-dependent cells could reemerge as the predominant population upon reculturing in a favorable environment.
* Corresponding author. t Present address: School of Microbiology, University of New South Wales, P.O. Box 1, Kensington, New South Wales 2033, Australia.
VOL. 28, 1990
Frozen Stock (-70 C)
I Test Test FIG. 1. Flow diagram showing the protocol for the passaging and testing of H. pylori for arachidonic acid resistance. ISA/BSA, Iso-Sensitest agar containing BSA.
The factor(s) responsible for the catalase independence is unknown. The ability of blood to prevent the accumulation of reactive elements such as superoxide and hydrogen peroxide appears responsible for the maintenance of a catalase-dependent population. The ability of BSA to afford some limited protection to these sensitive bacteria allows the development of the catalase-independent cells. Knapp and Melly (5) proposed that the bactericidal effects of arachidonic acid were mediated by a peroxidative process involving H202 and bacterial iron. They noted that not all bacteria were sensitive to arachidonic acid, with some gram-negative bacteria, such as Escherichia coli, being highly resistant. The most sensitive bacteria appeared to be the gram-positive ones, such as Staphylococcus aureus, and a proportion of gram-negative ones, such as Neisseria gonorrhoeae. Thus, differences in the structure of the cell envelope may contribute to arachidonic acid sensitivity. Examination of cell fractions of H. pylori by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal detectable differences between catalase-dependent and catalase-independent cells. We may conclude, therefore, either that there were differences in the nonprotein components of these cells or that fractionation of the bacteria and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were not sensitive enough to detect subtle differences in these cells. Another factor, the presence of endogenous catalase activity, did not appear to have an impact upon the sensitivity of H. pylori to arachidonic acid, as the isolates grown on various media were all catalase positive. However, the possibility remains that the catalase-independent cells are "leaky" with respect to their endogenous catalase and so could limit the peroxidation of arachidonic acid in their local environment. Such leaky cells would have a selective advantage over the catalase-dependent cells when cultured on Iso-Sensitest agar containing BSA and, after multiple passaging, would be the dominant population. Stuart L. Hazell is a C. J. Martin Fellow supported by the National Health and Medical Research Council of Australia. This work was supported in part by Veterans Affairs and in part by Public
2. 6. 4.
Blood ex BSA FIG. 2. Effect of arachidonic acid (0.01 mM, 1 h, room temperature) on the viability of isolates of H. pylori grown on blood agar (Blood) or Iso-Sensitest agar containing BSA (BSA) or following reculturing on blood agar after passaging on Iso-Sensitest agar containing BSA (Blood ex BSA). A, B, C, and D represent isolates 8801, 8802, 8803, and 8804, respectively. SD, Standard deviation. Health Service grant DK-39919 from the National Institute of Diabetes and Digestive and Kidney Diseases. LITERATURE CITED 1. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115:717-722. 2. Goodwin, C. S., J. A. Armstrong, T. Chilvers, M. Peters, M. D. Collins, L. Sly, W. McConnell, and W. E. S. Harper. 1989. Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylori comb. nov. and Helicobacter mustelae. comb. nov., respectively. Int. J. Syst. Bacteriol. 39:397-405. 3. Hazell, S. L., D. C. Markesich, D. J. Evans, Jr., D. G. Evans, and D. Y. Graham. 1989. Influence of media supplements on growth and survival of Campylobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis. 8:597-602. 4. Inouye, M., and J. P. Guthrie. 1969. A mutation which changes a membrane protein of E. coli. Proc. Natl. Acad. Sci. USA
64:957-961. 5. Knapp, H. R., and M. A. Melly. 1986. Bactericidal effects of polyunsaturated fatty acids. J. Infect. Dis. 154:84-94. 6. Marshall, B. J., and C. S. Goodwin. 1987. Revised nomenclature of Campylobacter pyloridis. Int. J. Syst. Bacteriol. 37:68. 7. Miles, A. A., and S. S. Misra. 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38:732-748.