Leyer & Johnson (1997)

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1997, p. 461–467 Vol. 63, No.

2
0099-2240/97/$04.0010
Copyright q 1997, American Society for Microbiology

Acid Adaptation Sensitizes Salmonella typhimurium to


Hypochlorous Acid
GREGORY J. LEYER1† AND ERIC A. JOHNSON1,2*
Departments of Food Microbiology and Toxicology1 and Bacteriology2,
University of Wisconsin, Madison, Wisconsin 53706
Received 15 July 1996/Accepted 6 November 1996

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

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and nonadapted salmonellae were investigated. Hypochlorous acid oxidized a higher percentage of cell surface
sulfhydryl groups in acid-adapted cells than in nonadapted cells, and sulfhydryl oxidation was correlated with
cell inactivation. HOCl caused severe metabolic disruptions in acid-adapted and nonadapted S. typhimurium,
such as respiratory loss and inability to restore the adenylate energy charge from a nutrient-starved state.
Sensitization of S. typhimurium to hypochlorous acid by acid adaptation also involved increased permeability
of the cell surface because nonadapted cells treated with EDTA became sensitized. The results of this study
establish that acid-adapted S. typhimurium cells are highly sensitized to HOCl oxidation and that inactivation
by HOCl involves changes in membrane permeability, inability to maintain or restore energy charge, and
probably oxidation of essential cellular components. This study provides a basis for improved practical
technologies to inactivate Salmonella and implies that acid pretreatment of food plant environments may
increase the efficacy of halogen sanitizers.

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

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Sulfhydryl determination. The cellular surface sulfhydryl concentration was acid-adapted or nonadapted cells were suspended in 0.1 M Tris-HCl buffer (pH
measured with 5,59-dithiobis(2-nitrobenzoic acid) (DTNB) (21, 48). Cells (10 ml 8.0) containing 10 mM EDTA. The cell suspensions were incubated for 1 or 2 h
of suspension) treated with HOCl for 10 min were quenched with 100 ml of 0.1 at 378C to permeabilize the outer membrane (49), washed twice with dH2O, and
M sodium thiosulfate, harvested by centrifugation, washed twice with an equal exposed to HOCl in PB for 10 min as previously described. Cell viability was
volume of dH2O, and resuspended in 5 ml of 0.1 M potassium phosphate buffer determined before and after treatments with Tris-EDTA and HOCl by plating
(pH 6.6) containing 1 mM MgSO4. The cell suspension was added to 10 ml of 0.1 on TPAP agar.
M Tris-HCl buffer (pH 8.0) containing 10 mM EDTA. To this suspension, 250 ml Statistical analysis. Each data point in the figures and tables is the mean of
of 10 mM DTNB in 0.1 M potassium phosphate buffer (pH 7.0) and 1.25 ml of triplicate experiments. Additionally, each set of conditions was replicated at least
10% (wt/vol) sodium dodecyl sulfate were added. The suspension was incubated once. The error bars in the figures represent one standard deviation from the
at 378C for 1 h, cooled to 08C, and centrifuged at 18,000 3 g for 10 min. The A412 mean.
of the supernatants was measured with a Response spectrophotometer (Gilford,
Oberlin, Ohio). A molar extinction coefficient of 13,600 for 5-thio-2-nitrobenzoic
acid was used (21). Results are presented as percent sulfhydryl concentration RESULTS
relative to untreated cells.
Respiration measurement. Respiration was estimated by measuring O2 con- Tolerance of S. typhimurium to chemical sanitizers. Acid-
sumption with a digital oxygen system oxygen electrode (Rank Brothers Ltd.,
adapted or nonadapted cells were treated with three classes of
Cambridge, England) and reported as a percentage of the original rate in un-
treated cells. Results are expressed as millimoles of O2 per hour per gram (dry sanitizers. The acid-adapted population survived significantly
cell weight) based on a concentration of 268 mM O2 in air-saturated buffer at better than nonadapted cells when challenged with 10 ppm of
258C (43). A 10-ml portion of an overnight culture was centrifuged and resus- an anionic acid sanitizer at pH 3.23 (Fig. 1A). After a 20-min
pended in 1.2 ml of PB (pH 6.0) and kept on ice until use. Then, 400 ml of cell exposure, a 1,000-fold difference in the cellular viability was
suspension was combined with 400 ml of 300 mM D-glucose in 3.2 ml of air-
saturated PB and held with occasional agitation at ambient temperature for 5 observed, and this differential remained throughout the incu-
min. After 5 min, respiration was monitored at ambient temperature by moni- bation, although both cell populations lost viability over time.
toring the oxygen electrode response on a Linear 1200 chart recorder (Linear Acid-adapted and nonadapted cells were also treated with
Instruments Corp., Reno, Nev.). Once an initial linear oxygen consumption rate an iodine-based sanitizer (Fig. 1B). Unexpectedly, acid adap-
was obtained, 20 ml of an appropriately diluted solution of HOCl was added to
the respiring suspension, and the subsequent oxygen consumption rate deter- tation sensitized the cells to the iodine sanitizer. The acid-
mined. adapted population died off extremely rapidly, losing 5 log
Determination of enzyme activities. The activities of various dehydrogenase units of an original 7-log-unit population during the first 10
enzymes in crude cell lysates from HOCl-treated or nontreated cells were de- min of exposure. After this time, the viable cell number re-
termined. The culture (225 ml, A600 ' 0.25) was centrifuged and resuspended in
1 ml of dH2O. The cell suspension was added to 224 ml of PB, treated with 1.125 mained relatively constant during the 30-min exposure, result-
ml of HOCl with stirring, and allowed to react for 10 min, and the reaction was ing in a ;1,000-fold difference in viability between the two cell
quenched by adding 200 ml of 0.1 M sodium thiosulfate. The cells were harvested populations.
by centrifugation at 12,000 3 g for 10 min, resuspended in 1 ml of 30 mM Acid-adapted populations of S. typhimurium were also mark-
Tris-HCl buffer (pH 8.0) containing 10 ml of 100 mM phenylmethylsulfonyl
fluoride, and passed twice through a French pressure cell (American Instrument edly sensitized to HOCl (Fig. 2A). As observed with cells
Co., Silver Spring, Md.) at 1,000 lb/in2. Whole cells and cell wall debris were treated with the iodophor, the acid-adapted cell population
removed by centrifugation at 12,000 3 g for 10 min, and crude cell lysates were experienced a rapid and marked viability loss during the initial
stored at 2808C until analysis. The activities of glyceraldehyde-3-phosphate exposure for 5 min, resulting in a ;1,000-fold difference in cell
dehydrogenase (GAPDH) (EC 1.2.1.12), lactate dehydrogenase (LDH) (EC
1.1.1.27), and malate dehydrogenase (MDH) (EC 1.1.1.37), were measured spec- viability between the two populations after 5 min. After this
trophotometrically by recording NADH oxidation (for LDH and MDH) or NAD time, the viabilities of both cell types remained relatively con-
reduction (for GAPDH) at 340 nm following substrate addition (8, 50). Gluta- stant. As expected, cell inactivation was dependent on the
mate dehydrogenase (GDH) activity (EC 1.4.1.2) was determined by measuring concentration of HOCl (Fig. 2B). At low HOCl levels, both cell
the NADPH oxidation rate following cell extract addition to a solution contain-
ing 2-ketoglutarate (6.5 mM), NADPH (0.15 mM), and NH4Cl (30 mM) in a
populations survived equally well. When the HOCl concentra-
1-ml final volume of Tris-HCl (30 mM) buffer (pH 8.0). Enzyme activities are tion was increased to 100 mmol/g of salmonellae, the acid-
expressed as units per milligram, where 1 U is defined as 1 nmol of NADPH adapted population was inactivated rapidly whereas the non-
(NADH) oxidized or NAD reduced per min. The protein concentration in all adapted population was inactivated much more slowly. Four
enzyme assays was 50 mg/ml.
EC measurements. Adenylate energy charge (EC) step-up in S. typhimurium
additional strains of Salmonella also exhibited the characteris-
was determined by quantifying extracted nucleotides from HOCl-treated or tic sensitization to HOCl after acid adaptation.
nontreated cells (9, 10). Acid-adapted or nonadapted cells (450 ml) were har- Oxidation of sulfhydryls on whole cells. HOCl is a powerful
vested by centrifugation and concentrated 10-fold in dH2O. The concentrated oxidant, and sulfhydryl groups of cell membranous compo-
cell suspension (4.5 ml) was added to 180 ml of 50 mM sodium phosphate buffer
(pH 6.0). Diluted HOCl (900 ml) was added, and the mixture was incubated at
nents are potential targets for oxidation and cell inactivation
ambient temperature for 15 min. The reaction was quenched by adding 200 ml of (20, 31, 46, 47). The sulfhydryl content of acid-adapted and
0.10 M sodium thiosulfate. HOCl-treated cells were centrifuged at 12,000 3 g for nonadapted cells was 94 and 121 nmol of sulfhydryl/mg (dry
VOL. 63, 1997 SENSITIZATION OF S. TYPHIMURIUM TO HOCl 463

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FIG. 2. Survival of acid-adapted (E) and nonadapted (F) S. typhimurium
during exposure to 475 mmol of HOCl/g (dry weight) of salmonellae (A) or to
various HOCl levels (B). Graph B shows HOCl dose-dependent killing kinetics.

with 355 mmol of HOCl/g, the adapted and nonadapted ex-


FIG. 1. Survival of acid-adapted (E) and nonadapted (F) S. typhimurium tracts retained approximately 52 and 74% of GAPDH activity,
during exposure to 10 ppm of anionic acid (pH 3.23) disinfectant (A) or 1.0 ppm
of iodine-based disinfectant (B). Error bars in this and subsequent figures indi-
respectively. When treated with various levels of HOCl, LDH
cate standard deviations determined from triplicate experiments; datum points activity initially increased and then declined in acid-adapted
with no error bars indicate that the error is smaller than the symbol. cells (Table 1). This pattern was observed repeatedly. In con-
trast, LDH was relatively stable in response to HOCl in non-
adapted cells. Higher activities of MDH were present in non-
adapted than in adapted cell extracts (Table 1). MDH was not
weight), respectively. Loss of sulfhydryl groups occurred at inactivated by the HOCl concentration examined, and MDH
approximately the same rate in both cell populations treated activity did not correlate with cell viability loss. GDH activity
with 190 mmol of HOCl/g of cells (Fig. 3). As the HOCl increased in both cell populations during exposure to increas-
concentration was increased, the sulfhydryls in acid-adapted
cells were lost more rapidly than were those in nonadapted
cells. At 475 mmol of HOCl/g of salmonellae, only 16% of the
original sulfhydryl groups in acid-adapted cells remained un-
oxidized after 1 h, whereas 61% of the sulfhydryl groups re-
mained in their reduced state in nonadapted cells. Sulfhydryl
group oxidation correlated with loss in cell viability.
Effect of HOCl on dehydrogenase enzyme activities. In the
experiment described in the previous section, acid-adapted
salmonellae showed a greater loss of sulfhydryls than did non-
adapted cells. Since oxidation of sulfhydryls has been suggested
as a mechanism of cell inactivation by chlorine, and since
glutamic acid and related primary metabolites have been im-
plicated in acid resistance (22, 27), the activities of selected
sulfhydryl dehydrogenase enzymes involved in primary metab-
olism were assayed after treatment with HOCl. The enzymes
examined varied in their sensitivity to HOCl. GAPDH was FIG. 3. Loss of cell surface sulfhydryl groups as a function of HOCl dose in
acid-adapted (E) and nonadapted (F) S. typhimurium. Data are given as the
inactivated by HOCl to a greater degree in adapted than in percentage of the original sulfhydryl groups remaining, with 100% representing
nonadapted cells (Table 1). This pattern of enzyme inactiva- 121 nmol of sulfhydryl/mg (dry weight) in nonadapted cells and 94 nmol of
tion correlated with loss of viability (Fig. 2B). When treated sulfhydryl/mg (dry weight) in acid-adapted cells.
464 LEYER AND JOHNSON APPL. ENVIRON. MICROBIOL.

TABLE 1. Sulfhydryl-dependent dehydrogenase inactivation by HOCl in acid-adapted and nonadapted S. typhimurium


Activity (U/mg) ofa:
HOCl concn
GAPDH LDH MDH GDH
(mmol/g of salmonellae)
Acid adapted Nonadapted Acid adapted Nonadapted Acid adapted Nonadapted Acid adapted Nonadapted

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

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ATPase enzymes have been shown to be involved in ATP cells have a decreased ability to maintain and step up their EC
synthesis and maintenance (39) and have been implicated in when treated with HOCl.
the acid tolerance response in S. typhimurium (40). Therefore, Inhibition of O2 uptake by HOCl. Since acid-adapted cells
we investigated the levels of adenylate nucleotides by deter- were impaired in energy production and maintenance, we in-
mining the adenylate EC in acid-adapted and nonadapted vestigated oxygen uptake in adapted and nonadapted cell pop-
cells. The adenylate EC and its recovery from energy starvation ulations. HOCl treatment affected oxygen uptake differently in
have been used to assess the metabolic well-being of cells (6, 9, acid-adapted and nonadapted S. typhimurium. Oxygen uptake
11, 18). Healthy bacteria can increase their EC within minutes was more strongly inhibited by HOCl in acid-adapted S. typhi-
from a resting-stage EC of 0.1–0.2 to 0.8–0.9 when given an murium cells than in nonadapted cells (Fig. 5). Immediately
appropriate energy source (9). The ability of acid-adapted and after HOCl addition, an abrupt decline in O2 uptake was ob-
nonadapted S. typhimurium cells to undergo an EC step-up served. When acid-adapted cells were treated with 150 mmol of
after exposure to various HOCl levels was determined (Fig. 4). HOCl/g, the oxygen uptake rate decreased to approximately
Energy-starved cells maintained an EC of 0.10 to 0.25. Un- 10% of the original level. At the same HOCl concentration, the
treated nonadapted and acid-adapted S. typhimurium cells nonadapted population retained approximately 50% of the
were able to rapidly achieve an EC of .0.60. The ability to original oxygen uptake rate. Other concentrations of HOCl
produce an EC step-up decreased markedly with increased also differentially affected the O2 uptake in acid-adapted and
exposure to HOCl in both adapted and nonadapted cells, and nonadapted cell populations (Fig. 5). Hence, acid-adapted cell
adapted cells were less able to rapidly restore the energy populations were impaired in oxygen uptake compared to non-
charge. When treated with 77 mmol of HOCl/g, corresponding adapted cells on treatment with HOCl.
to a ;30% viability loss in adapted and nonadapted cells, the Outer membrane disruption and HOCl tolerance. We pre-
acid-adapted cells stepped up their EC from 0.13 to 0.27 in 40 viously demonstrated that cell surface characteristics are al-
min whereas the nonadapted cells stepped up to an EC of 0.55 tered in acid-adapted S. typhimurium (32), and therefore we
in 20 min. The inability of acid-adapted S. typhimurium to investigated whether disrupting the outer membrane by EDTA
restore their EC was even more pronounced when the HOCl treatment (49) would enhance the sensitivity of nonadapted
concentration was increased to 191 mmol/g, at which the via- cells to HOCl. Untreated nonadapted and adapted cell popu-
bility of acid-adapted cells was reduced to 0.03% of the original lations displayed the characteristic ;10,000-fold difference in
level and cells were unable to increase their EC above the 0.22 sensitivity to HOCl (Fig. 6). The difference in sensitivity was
decreased after treatment with Tris-EDTA. After 1 h of treat-
ment with Tris-EDTA, the difference in sensitivity was only

FIG. 4. Inhibition of EC step-up by HOCl oxidation. The EC step-up was


measured after HOCl oxidation and energy starvation and was restored by the FIG. 5. Inhibition of oxygen uptake by HOCl in acid-adapted (E) or non-
addition of 222 mM glucose at time zero. Open symbols represent acid-adapted adapted (F) S. typhimurium. The data represented are the result of three deter-
S. typhimurium, and solid symbols represent nonadapted cells. E, F, 0 mmol of minations in which 100% control was 7.0 to 9.7 mmol of O2/h/g (dry weight) in
HOCl/g of salmonellae; h, ■, 77 mmol of HOCl/g; Ç, å, 191 mmol of HOCl/g. nonadapted cells and 6.4 to 9.7 mmol of O2/h/g (dry weight) in acid-adapted cells.
VOL. 63, 1997 SENSITIZATION OF S. TYPHIMURIUM TO HOCl 465

chromosomal DNA replication (42). The bactericidal activity


of HOCl has not, to our knowledge, been hypothesized to
depend on disruption of membrane permeability (1, 36) or
inhibition of protein synthesis (36). In this study, we evaluated
the inactivation of several cellular targets by HOCl and at-
tempted to identify targets which were disrupted prior to cell
death and therefore would contribute to loss of viability.
Cell surface sulfhydryl oxidation correlated with loss in the
cell viability of adapted and nonadapted S. typhimurium, but
nonadapted cells were more resistant on extended exposure.
The difference in the resistance of nonadapted and adapted
cells to HOCl may be related to the overall sulfhydryl levels in
the two populations. Nonadapted cells had a greater quantity
of sulfhydryl groups than did acid-adapted cells, which may
have bestowed greater protection. To assess the role of sulf-
hydryl oxidation in cell killing, we also evaluated the inactiva-
tion of NAD(P)-linked sulfhydryl enzymes by HOCl. The

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NAD(P)-linked enzymes examined in this study differed in
their activities in acid-adapted and nonadapted cells and also
in their susceptibility to HOCl. Inactivation of GAPDH cor-
FIG. 6. Sensitization of S. typhimurium to HOCl after disruption of the outer
membrane by EDTA. The abscissa represents the length of incubation in the related with loss in cell viability. GADPH contains three sulf-
presence of 10 mM EDTA in 0.1 M Tris-HCl buffer (pH 8.0). Cells were exposed hydryl groups in the catalytic site, one of which is essential for
to 475 mmol of HOCl/g of salmonellae for 10 min before viability determination. activity and is also exceptionally reactive with HOCl (3). LDH
activity declined during treatment with HOCl, but the decline
did not correlate with loss of cell viability. The other two
;10-fold. After 2 h of exposure to Tris-EDTA, the nonadapted enzymes examined, MDH and GDH, were not inactivated by
cells had greater sensitivity to HOCl than the acid-adapted HOCl. GDH activity increased with increasing dose of HOCl
cells did. in both acid-adapted and nonadapted cells. Rakita et al. (41)
also reported that GDH activity increased on incubation with
an activated myeloperoxidase antimicrobial system. These
DISCUSSION changes may indicate that changes in the redox potential could
increase the activity or synthesis of various enzymes. Since we
The ability of microbial pathogens to adapt to environmen-
did not observe an obvious correlation between the inactiva-
tal stresses such as acid contributes to their survival in the
tion of metabolic enzymes, we investigated the role of energy
natural environment. Acid adaptation probably plays an im-
maintenance and oxygen uptake in promoting survival in
portant role in food safety, since many foods depend on acidic
conditions for preservation (16). We previously showed that HOCl-treated cells.
acid-adapted S. typhimurium and Escherichia coli O157:H7 sur- Previous studies indicated that abolition of ATP formation
vived better in certain acidic and fermented foods than did contributes to bacterial killing by HOCl (9–11). In E. coli,
their nonadapted counterparts (33, 34). Acid-adapted S. typhi- HOCl-promoted inactivation was accompanied by inhibition of
murium cells have an enhanced ability to maintain their inter- respiration (2, 43). Mutations in the genes in the atp operon
nal pH (pHi) at or near a neutral level when exposed to very also have been shown to abolish the acid tolerance response in
acidic conditions (pH , 4.0) (23). Therefore, it was not sur- S. typhimurium (22, 40). In our studies, respiration was inacti-
prising that acid adaptation enhanced their tolerance to an vated by HOCl rapidly in acid-adapted S. typhimurium but
anionic acid disinfectant. However, it was surprising that acid- more slowly in nonadapted cells. Several essential bacterial
adapted S. typhimurium cells exhibited a dramatic (;10,000- respiratory components including porphyrins, heme, heme
fold) sensitization to halogen sanitizers. In this study, we in- proteins, Fe-sulfur proteins, and cytochromes are sensitive to
vestigated the physiological mechanisms contributing to the oxidation by HOCl (3, 44). Since respiration was decreased
unexpected differential sensitivity to HOCl in acid-adapted prior to cell death, its loss could contribute to inactivation, and
and nonadapted S. typhimurium. our data support the hypothesis that maintenance of respira-
HOCl is an effective disinfectant partly because most micro- tory ability is especially important for acid-adapted cells ex-
organisms do not possess specific enzymatic mechanisms for posed to HOCl.
detoxification of HOCl, as they do for other oxidants such as The adenylate nucleotide charge level in bacteria is tightly
reactive oxygen species. The mechanism of inactivation of mi- regulated and is important in maintaining cell viability during
crobial cells by HOCl is not completely elucidated but is be- energy starvation (18). HOCl has previously been demon-
lieved to involve the oxidation of critical cellular components strated to impair the ability of E. coli to maintain an adenylate
and depletion of cellular energy. HOCl generated by the ac- energy charge required for viability (9, 11). Our data support
tivity of myeloperoxidase, H2O2, and a halide provides an the hypothesis that a useful index of the metabolic well-being
effective microbiocidal system for mammalian cells (29). Re- of cells during treatment with HOCl is the ability of cells to
cently, acidification of hypochlorite solutions has been shown maintain a suitable EC attainable or to step up the EC when
to generate ozone, a strong oxidant (30). HOCl causes destruc- presented with nutrients (9, 11). The difference in the ability of
tion of cellular components by several mechanisms: (i) sulfhy- acid-adapted or nonadapted S. typhimurium cells to step up the
dryl oxidation (46, 47), (ii) inactivation of iron-sulfur centers EC after treatment with HOCl indicated that acid-adapted
(3, 28, 44, 45), (iii) inactivation of enzymes involved in respi- cells were severely impaired in their ability to regenerate en-
ration (2, 41), (iv) disruption of nutrient transport (1, 9), (v) ergy stores. This conclusion is in accordance with the reports
inhibition of energy generation (9, 11), and (vi) inactivation of that mutations in the genes encoding Mg21-dependent proton-
466 LEYER AND JOHNSON APPL. ENVIRON. MICROBIOL.

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

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In summary, this communication has demonstrated that ac-
and yeast. Adv. Microb. Physiol. 15:253–306.
id-adapted S. typhimurium is highly sensitized to HOCl action. 19. Cords, B. R., and G. R. Dychdala. 1993. Sanitizers: halogens, surface-active
Acid-adapted salmonellae possess certain fundamental changes agents, and peroxides. In P. M. Davidson and A. L. Branen (ed.), Antimi-
in metabolism that predispose them to inactivation. Most im- crobials in foods, 2nd ed. Marcel Dekker, Inc., New York, N.Y.
portantly, the inability to generate ATP through respiration 20. Dychdala, G. R. 1991. Chlorine and chlorine compounds, p. 131–151. In S. S.
Block (ed.), Disinfection, sterilization, and preservation, 4th ed. Lea &
and to maintain an energy level necessary for survival is im- Febiger, Philadelphia, Pa.
portant. Also, outer membrane permeability and structure are 21. Ellman, G. L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:
altered in acid-adapted S. typhimurium. This study provides a 70–77.
basis for the development of practical methods to increase the 22. Foster, J. W. 1995. Low pH adaptation and the acid tolerance response of
Salmonella typhimurium. Crit. Rev. Microbiol. 21:215–237.
efficacy of halogen sanitizers, such as pretreating the food plant 23. Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid
environment with a mild acid prior to sanitization. Our study tolerance response of Salmonella typhimurium. J. Bacteriol. 173:5129–5135.
also suggests that it may be feasible to reduce the quantities of 24. Foster, J. W., and H. K. Hall. 1990. Adaptive acidification tolerance response
halogen sanitizers used industrially and thereby decrease the of Salmonella typhimurium. J. Bacteriol. 172:771–778.
25. Gerhardt, P. 1981. Diluents and biomass measurement, p. 504–507. In P.
possible deleterious impact of chlorine and related compounds Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R.
on human health. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology.
American Society for Microbiology, Washington, D.C.
ACKNOWLEDGMENTS 26. Gribble, G. W. 1995. Chlorine and health. American Council on Science and
Health, New York, N.Y.
We thank Marty Krause for excellent technical assistance with ex- 27. Hersh, B. M., F. T. Farooq, D. N. Barstad, D. L. Blankenhorn, and J. L.
periments. Slonczewski. 1996. A glutamate-dependent acid resistance gene in Esche-
This research was supported by contributions by the food industry to richia coli. J. Bacteriol. 178:3978–3981.
the Food Research Institute, by the Southeastern Poultry Association, 28. Hurst, J. K., W. C. Barrette, Jr., B. R. Michel, and H. Rosen. 1991. Hypo-
by the Wisconsin Center for Dairy Research through Dairy Manage- chlorous acid and myeloperoxidase-catalyzed oxidation of iron-sulfur clus-
ters in bacterial respiratory dehydrogenases. Eur. J. Biochem. 202:1275–
ment Inc. and the Wisconsin Milk Marketing Board, by NRI Compet-
1282.
itive Grants Program/USDA (94-37201-1026), and by the College of 29. Hurst, J. K., and W. C. Barrette, Jr. 1989. Leukocytic oxygen activation and
Agriculture and Life Sciences, University of Wisconsin, Madison. microbicidal oxidative toxins. Crit. Rev. Biochem. Mol. Biol. 24:271–328.
30. Khan, A. U., and M. Kasha. 1994. Singlet molecular oxygen evolution upon
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