Leyer & Johnson (1997)
Leyer & Johnson (1997)
Leyer & Johnson (1997)
2
0099-2240/97/$04.0010
Copyright q 1997, American Society for Microbiology
Acid adaptation of Salmonella typhimurium at a pH of 5.0 to 5.8 for one to two cell doublings resulted in
marked sensitization of the pathogen to halogen-based sanitizers including chlorine (hypochlorous acid) and
iodine. Acid-adapted S. typhimurium was more resistant to an anionic acid sanitizer than was its nonadapted
counterpart. A nonselective plating medium of tryptose phosphate agar plus 1% pyruvate was used throughout
the study to help recover chemically stressed cells. Mechanisms of HOCl-mediated inactivation of acid-adapted
Salmonella spp. are the major cause of foodborne disease in The food, medical, water, and environmental industries
the United States and certain other countries. It has been commonly use chemical sanitizers to eradicate pathogenic and
estimated that 6.5 to 33 million people become ill from con- spoilage microorganisms from foods, medical devices, and the
taminated food annually in the United States, resulting in an environment. Chlorine, in its various forms, is the most widely
estimated 6,000 deaths and an estimated economic cost of $2.5 used of all chemical sanitizers (13, 14, 19). Hypochlorites are
billion to $3.4 billion per year (5). Of these illnesses, about half especially useful because of their rapid activity against a wide
have been reported to be caused by salmonellae (12). A variety variety of microorganisms and their low cost. However, con-
of foods have been implicated in salmonellosis, including fer- troversy has arisen in recent years regarding the potential del-
mented foods and acidic products (12). Despite extensive con- eterious health effects of chlorine and its by-products (4, 13,
trol efforts by regulatory agencies and the food industry, the 26). This study was undertaken to evaluate the efficacy and
percentage of outbreaks due to Salmonella is increasing at a mechanisms of commonly used chemical disinfectants against
rate greater than that of outbreaks due to several other bac- S. typhimurium that had previously been exposed to a mild acid
terial pathogens (12). stress. Unexpectedly, our results indicate that acid adaptation
Salmonellae are widespread in nature and are commonly strongly sensitizes salmonellae to hypochlorous acid.
found as inhabitants of animal intestinal tracts. By virtue of
their ubiquity, Salmonella spp. are exposed to a variety of
chemical and environmental stresses. Salmonellae and other MATERIALS AND METHODS
bacteria can adapt to stresses by regulating the expression of
Chemicals and reagents. All chemicals were obtained from Sigma Chemical
specific sets of genes termed stimulons (38). Salmonellae and Co., St. Louis, Mo. Tryptic soy broth was purchased from Difco, Detroit, Mich.
other species of the family Enterobacteriaceae are able to ex- Bacterial strains and growth conditions. S. typhimurium LT2 (provided by
press adaptive responses that allow survival under acid con- Laszlo Csonka, Purdue University) was used throughout this study, and the
ditions (35). Acid adaptation in Salmonella typhimurium procedures for growth and acid adaptation in medium E were the same as
previously described (32, 33). Four additional strains of Salmonella (KB17,
enhances the ability to withstand severe acid stress by main- SL1917, SH1906, and 7454) were also tested for HOCl sensitization after acid
taining internal pH homeostasis (19, 20), 24. In addition to adaptation. For acid adaptation, S. typhimurium was grown statically in medium
withstanding severe acid stress in media, acid-adapted S. typhi- E (pH 7.6) with 0.4% glucose until the culture reached an absorbance at 600 nm
murium showed increased survival in acidic and fermented (A600) of ;0.1, at which time the medium was acidified to pH 5.0 to 5.8 with a
small volume of 10 N HCl. The cells were grown to an A600 of 0.25 to 0.30. The
foods (33, 34), and exhibited cross-protection against various nonadapted control cells were grown to an A600 of 0.25 to 0.30 in nonacidified
environmental stresses (32). medium E. Then 1 ml each of the acid-adapted and nonadapted cells was
harvested by centrifugation, washed once in 1 ml of distilled water (dH2O), and
resuspended in 100 ml of dH2O. These cells were used to determine resistance to
* Corresponding author. Mailing address: Department of Food Mi- chemical disinfectants. Cellular viability after various treatments was determined
by plating onto nonselective tryptose phosphate agar–0.1% pyruvate (33) to
crobiology and Toxicology, University of Wisconsin, 1925 Willow Dr.,
allow rescusitation of chemically stressed cells.
Madison, WI 53706. Phone: (608) 263-7944. Fax: (608) 263-1114. E- Dry weight and protein determination. Bacterial dry weights were determined
mail: eajohnso@facstaff.wisc.edu. with 10-fold-concentrated cell suspensions. A 1-ml portion of the concentrated
† Present address: Abbott Laboratories, Ross Products Division, cell suspension was dried in an aluminum dry pan with a 55-mm fiberglass filter
Columbus, OH 43219. for 16 to 24 h at 1058C until a constant weight was obtained (25). Protein was
461
462 LEYER AND JOHNSON APPL. ENVIRON. MICROBIOL.
determined by using the bicinchoninic acid protein assay reagent purchased from 10 min, resuspended in 4.5 ml of E buffer (medium E without glucose supple-
Pierce, Rockford, IL. mentation), and incubated for 30 min at 378C with shaking to starve the cells for
Sanitizer preparation. Chemical sanitizers were provided by the Diversey energy. After a 30-min starvation, 500 ml was sampled for determination of
Wyandotte Corp., Wyandotte, Mich. The liquid chlorinated sanitizer contained adenylate nucleotide concentrations. Then 400 ml of a 2.22 M D-glucose solution
9.2% (wt/vol) sodium hypochlorite as its active ingredient. The hypochlorite was added to the remaining cell suspension, and the cells were incubated at 378C
stock solution was routinely titrated by the available-chlorine method (17). The with shaking and sampled at time intervals for nucleotide concentrations.
anionic acid sanitizer contained 30% (wt/vol) phosphoric acid as its active ingre- Nucleotide concentrations were measured chromatographically by analyzing S.
dient. The iodine-based disinfectant contained organic complexes of iodine, typhimurium perchloric acid extracts. Cell suspension (500 ml) was added to 200
providing 1.75% titratable iodine. The iodine-based and anionic acid disinfectant ml of ice-cold 3 M perchloric acid. The acid-quenched solution was allowed to
stock solutions were prepared as specified by the manufacturer. Liquid chlori- stand on ice for 30 min, centrifuged at 12,000 3 g for 5 min, and neutralized with
nated sanitizer dilutions were based on available-chlorine titration results. 200 ml of 3 M KOH and 100 ml of 50 mM potassium phosphate (pH 3.0). The
Tolerance of S. typhimurium toward chemical disinfectants. The tolerance of precipitated perchlorate ions were removed by centrifugation, and the superna-
S. typhimurium to sanitizers was determined at ambient temperature by adding tant was filtered and stored at 2808C until chromatographic analysis. Nucleotide
100 ml of cell suspension to 4.0 ml of dH2O (for anionic acid and iodine concentrations were quantified on a high-pressure liquid chromatography Hy-
sanitizers) or to 4.0 ml of 50 mM sodium phosphate buffer (PB), (pH 6.0) (for dropore-AX anion-exchange column (Rainin Instruments Co., Inc., Woburn,
HOCl studies). Experiments with HOCl were performed with PB at pH 6.0, at Mass.). The solvent system that gave optimum resolution was essentially the
which approximately 94% of HOCl is in its nondissociated state (20). The HOCl same as that described by Brown et al. (15). The initial eluant was 5 mM KH2PO4
concentration is expressed as micromoles of HOCl per gram (where 475 mmol is (pH 3.0) (A), and the final eluant was 500 mM KH2PO4 (pH 4.0) (B). A linear
approximately equivalent to 0.5 ppm HOCl). Viability was determined immedi- gradient from 100% A to 100% B was run over 40 min with a flow rate of 1.0
ately prior to and at appropriate time intervals after addition of the sanitizers. ml/min. The EC of the adenylate system (7) is defined as follows: ([ATP] 1 0.5
Samples were diluted in tryptic soy broth to neutralize residual sanitizer (37) [ADP])/([ATP] 1 [ADP] 1 [AMP]).
prior to plating. Outer membrane permeabilization by EDTA and HOCl tolerance. Washed
0 1.921 (0.165) 1.981 (0.127) 0.057 (0.019) 0.065 (0.029) 1.462 (0.026) 3.237 (0.068) 0.029 (0.002) 0.131 (0.005)
119.2 1.875 (0.08) 1.747 (0.145) 0.119 (0.006) 0.036 (0.004) 1.501 (0.081) 3.705 (0.145) 0.689 (0.054) 0.332 (0.019)
238.4 1.131 (0.17) 1.641 (0.087) 0.066 (0.004) 0.039 (0.014) 1.603 (0.037) 3.457 (0.095) 1.499 (0.044) 0.521 (0.008)
357.6 1.032 (0.13) 1.393 (0.074) 0.013 (0.012) 0.033 (0.009) 1.401 (0.088) 3.422 (0.109) 1.01 (0.061) 0.843 (0.052)
a
Standard deviations of triplicate determinations are given in parentheses.
ing amounts of HOCl (Table 1). These results suggested that original charge. When the same dose was given to nonadapted
the inactivation of sulfhydryl enzymes by HOCl was dependent cells, the viability was reduced to 2.8% of the original level but
on the particular enzyme and may contribute to cell killing. the cells were still able to step up the EC from 0.16 to 0.44.
EC step-up in HOCl-treated S. typhimurium. Ion-motive These results clearly show that acid-adapted energy-starved
translocating ATPase abolish acid tolerance in S. typhimurium 11. Barrette, W. C., Jr., J. M. Albrich, and J. K. Hurst. 1987. Hypochlorous
(40). acid-promoted loss of metabolic energy in Escherichia coli. Infect. Immun.
55:2518–2525.
The dramatic sensitization of acid-adapted cells to HOCl 12. Bean, N. H., and P. M. Griffin. 1990. Foodborne disease outbreaks in the
may not be entirely explained by differences in their suscepti- United States, 1973–1987: pathogens, vehicles and trends. J. Food Prot.
bility to HOCl inactivation of sulfhydryls, ATP production, and 53:807–814.
energy maintenance. Our data suggest that the ability of HOCl 13. Blatchley, E. R. III. 1994. Disinfection and antimicrobial processes. Water
Environ. Res. 66:361–368.
to penetrate through the outer membrane may also be impor- 14. Block, S. S. (ed.). 1991. Disinfection, sterilization and preservation, 4th ed.
tant in sensitization. We previously showed that the outer Lea & Febiger, Philadelphia, Pa.
membrane of acid-adapted S. typhimurium has a different com- 15. Brown, E. G., R. P. Newton, and N. M. Shaw. 1982. Analysis of free nucle-
position than that of nonadapted cells (32). In this study, sen- otide pools of mammalian tissues by high-pressure liquid chromatography.
Anal. Biochem. 123:378–388.
sitization of nonadapted cells was obtained by treatment with 16. Brown, M. H., and I. R. Booth. 1991. Acidulants and low pH, p. 22–43. In
EDTA, which is known to disrupt outer membrane permeabil- N. J. Russell and G. W. Gould (ed.), Food preservatives. Blackie, Glasgow,
ity. The actual porin or channel which allows HOCl penetra- Scotland.
tion in gram-negative cells has not been identified. It would be 17. Casse, R. A., R. L. Bradley, Jr., and R. R. Williams. 1985. Chemical and
physical methods: available chlorine. In G. H. Richardson (ed.), Standard
valuable to identify the channels involved, since this would methods for the examination of dairy products, 15th ed. American Public
advance our knowledge of the mechanisms of inactivation of Health Association, Washington, D.C.
salmonellae and other pathogens by halogen sanitizers. 18. Chapman, A. G., and D. E. Atkinson. 1977. Adenine nucleotide concentra-
tions and turnover rate. Their correlation with biological activity in bacteria
properties, and significance to cell function. Trends Biochem. Sci. 12:146– 45. Rosen, H., and S. J. Klebanoff. 1982. Oxidation of Escherichia coli iron
150. centers by the myeloperoxidase-mediated microbicidal system. J. Biol.
40. Portillo, F. G., J. W. Foster, and B. B. Finlay. 1993. Role of acid tolerance Chem. 257:13731–13735.
response genes in Salmonella typhimurium. Infect. Immun. 61:4489–4492. 46. Thomas, E. L. 1979. Myeloperoxidase, hydrogen peroxide, chloride antimi-
41. Rakita, R. M., B. R. Michel, and H. Rosen. 1990. Differential inactivation of crobial system: nitrogen-chlorine derivatives of bacterial components in bac-
Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated tericidal action against Escherichia coli. Infect. Immun. 23:522–531.
antimicrobial system. Biochemistry 29:1075–1080. 47. Thomas, E. L. 1979. Myeloperoxidase-hydrogen peroxide-chloride antimi-
42. Rosen, H., J. Orman, R. M. Rakita, R. Michel, and D. R. VanDevanter. 1990.
crobial system: effect of exogenous amines on antibacterial action against
Loss of DNA-membrane interactions and cessation of DNA synthesis in
Escherichia coli. 25:110–116.
myeloperoxidase-treated Escherichia coli. Proc. Natl. Acad. Sci. USA 87:
10048–10052. 48. Thomas, E. L., and T. M. Aune. 1978. Oxidation of Escherichia coli sulfhydryl
43. Rosen, H., R. M. Rakita, A. M. Waltersdorph, and S. J. Klebanoff. 1987. components by the peroxidase-hydrogen peroxide-iodide antimicrobial sys-
Myeloperoxidase-mediated damage to the succinate oxidase system of Esch- tem. Antimicrob. Agents Chemother. 13:261–265.
erichia coli. J. Biol. Chem. 242:15004–15010. 49. Vaara, M. 1992. Agents that increase the permeability of the outer mem-
44. Rosen, H., and S. J. Klebanoff. 1985. Oxidation of microbial iron-sulfur brane. Microbiol. Rev. 56:395–411.
centers by the myeloperoxidase-H202-halide antimicrobial system. Infect. 50. Worthington, C. C. (ed.). 1988. Worthington enzyme manual. Worthington
Immun. 47:613–618. Biochemical Corp., Freehold, N.J.