Lactoperoxidase-Thiocyanate-Hydrogen PeroxideSystem

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The deep rough mutant TA1535, with the most permeable cell envelope, was killed .... later mutations impart a rough character to the cell wall and increase the ...

INFECTION

AND

IMMUNITY, Mar. 1983, p. 1187-1195

Vol. 39, No. 3

0019-9567/83/031187-09$02.00/0 Copyright © 1983, American Society for Microbiology

Effect of Growth Phase and Cell Envelope Structure on Susceptibility of Salmonella typhimurium to the Lactoperoxidase-Thiocyanate-Hydrogen Peroxide System MICHAEL A. PURDY, JORMA TENOVUO, KENNETH M. PRUITT,* AND WILLIAM E. WHITE, JR.t Department ofBiochemistry, University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294 Received 19 August 1982/Accepted 29 November 1982

The lactoperoxidase-thiocyanate-hydrogen peroxide system was found to have both bacteriostatic and bactericidal activities against strains of Salmonella typhimurium. The bactericidal activity was clearly dependent on the permeability of the bacterial cell envelope. The deep rough mutant TA1535, with the most permeable cell envelope, was killed both at neutral and acid pH, whereas very little or no killing was observed with the intact cells of the parent strain hisG46. The Agal mutant, TA1530, representing an intermediate in cell envelope permeability, was inhibited to a much lesser extent than TA1535. Bacteria in log phase of growth were more sensitive to the bactericidal effects than were those in stationary phase. Growth phase had little influence on the bacteriostatic effects. The hisG46 strain produced significant quantities of acid in the presence of glucose. This acid production was inhibited by the lactoperoxidase-thiocyanatehydrogen peroxide system, and, in contrast to results obtained with several strains of streptococci, this inhibition was not reversed by addition of a reducing agent (2-mercaptoethanol). Peroxidase enzymes, thiocyanate ions (SCN-), and hydrogen peroxide (H202) provide an antimicrobial system in human saliva and bovine milk (6, 11, 23, 30). Human milk also contains peroxidase activity, but this activity is derived from milk leukocytes and is probably due to myeloperoxidase (15). These peroxidases catalyze the oxidation of SCN- to hypothiocyanite (OSCN-) (2, 8), which oxidizes protein sulfhydryl groups to sulfenyl-thiocyanate derivatives (36). The antibacterial effect of these reactions can to some extent be reversed by sulfhydryl compounds such as dithiothreitol, 2mercaptoethanol, and glutathione, which reduce both the sulfenyl derivatives and excess OSCN(19, 30, 33, 36). Although numerous studies have been published on the antimicrobial mechanism of the lactoperoxidase (LP) system (LP-SCN--H202) against both gram-positive and gram-negative bacteria, the results are still controversial with regard to the bacteriostatic-bactericidal action of the system (13, 22, 34, 35). Cell envelope permeability has been suggested to be an important factor in determining the susceptibility of Escherichia coli to the LP system (34) or to the myeloperoxidase-Cl--H202 system (28). The t Present address: Toxicology Branch, Chemical Systems Laboratory, Aberdeen Proving Ground, MD 21010.

present study was designed to determine the role of cell envelope permeability in the bacteriostatic-bactericidal action of the LP system against three different strains of Salmonella typhimurium with different degrees of cell envelope permeability. In addition, the effect of bacterial growth phase on susceptibility to the LP system was studied. Because of the recent observation that it is possible to increase the concentration of OSCN- in human saliva in vivo (21), it is of considerable interest to know the susceptibility of various bacteria, especially pathogens, to this naturally occurring defense mechanism. Remarkable improvement in the preservation of milk under field conditions by the enhancement of the bovine milk LP system has also been reported (5). Furthermore, calves fed with milk which was supplemented with LP system components show considerably better live-weight gains than calves receiving unsupplemented milk (24). Thus, the enhancement of the LP system seems to offer attractive possibilities for preventing the growth and the harmful effects of various pathogens. MATERIALS AND METHODS Chemicals. LP, purified from bovine milk, was obtained from Sigma Chemical Co., St. Louis, Mo. The purity index (absorbance at 412 nm/absorbance at 280

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PURDY ET AL.

nm) of the preparation was 0.82. Nbs2 (5,5'-dithiobis2-nitrobenzoic acid) was a product of Aldrich Chemical Co., Milwaukee, Wis. Nbs2 was reduced to Nbs (5thio-2-nitrobenzoic acid) immediately before use by addition of 2-mercaptoethanol. The Nbs concentration was calculated from the molar extinction coefficient of 13,600 at 412 nm (7). Potassium thiocyanate was a product of Sigma, and H202 was obtained from Fisher Scientific Co., Springfield, N.J., as a 30% solution. Catalase (from bovine liver) was purchased from Sigma. Unless otherwise stated, all of the reactions were carried out in phosphate-buffered saline (0.O1M potassium phosphate in saline, pH 6.5). Salmonella strains. S. typhimurium hisG46, TA1530, and TA1535 were obtained from B. N. Ames, University of California, Berkeley. These strains were originally developed as tester strains for mutagenicity and carcinogenicity (1). They are all carrying the hisG46 mutation. All three strains have defects in the uvrB gene which render them incapable of excision repair. In addition, strain TA1530 contains a Agal mutation, and strain TA1535 contains an rfa- mutation. These later mutations impart a rough character to the cell wall and increase the permeability of the cell envelope. The hisG46 strain (the parent strain of TA1530 and TA1535) has a normal cell envelope and is not permeable to crystal violet (1), the triphenyl triamine that is used to distinguish gram-positive from gram-negative bacteria. The cell envelope of strain TA1530 is more permeable than that of hisG46, but TA1530 is also not sensitive to crystal violet. However, the lipopolysaccharide defects in the cell envelope of strain TA1535 render the cell envelope of that organism permeable to crystal violet. Bactericidal and bacteriostatic effects of the LP system. In one series of experiments, the effect of pH and of generation of OSCN- in the presence of bacteria were evaluated. Bacteria (108 CFU/ml) were added to the medium after the pH had been adjusted by addition of HCI. Solutions of components of the LP system were then added to the mixture in the following order (calculated final concentrations are given in parentheses): SCN- (10 mM), LP (2.5 ,ug/ml), and H202 (0.7 mM), and the mixture was incubated for 1 h at 37°C. Viability was determined by placing an appropriate dilution of the culture being tested onto nutrient broth plates (triplicate samples) containing 1.5% agar (Difco Laboratories, Detroit, Mich.). The plates were counted after incubating overnight at 37°C. OSCN- concentrations were measured 3 to 10 min after H202 addition. For controls, only H202 was added. The effect on growth of generation of OSCN- in the of the bacteria was examined as follows. The strain to be studied was incubated overnight at 37°C in Vogel-Bonner (VB) salts (37), supplemented with histidine (30 ,ug/ml), biotin (2 ,ug/ml), and glucose (2%). The overnight culture was diluted with VB salts (plus glucose) medium to give an absorbance at 600 nm of 0.1 (corresponding to 108 CFU/ml); final volume of the mixture was 50 ml. The components of the LP system were added to the mixture immediately after the addition of bacteria. (The final calculated concentrations of the various components are given in the legends to Fig. 2 and 3.) In all experiments, H202 was added slowly (in small portions during a 5-min period) to avoid possible toxicity by unreacted H202 toward bacteria. The growth of these cultures was followed by presence

INFECT. IMMUN.

removing 1-ml aliquots at various times and measuring the absorbance at 600 nm with a Cary 219 spectrophotometer. (As a matter of convenience over the course of these studies, two different types of spectrophotometers were used for the absorbance measurements. There were no essential differences between results with the two different techniques.) An overnight culture of either hisG46 or TA1535 grown in nutrient broth (8 g of nutrient broth [Difco] and 4.5 g of NaCl per liter) was diluted 1:20 in fresh nutrient broth to a final volume of 10 ml. This dilution constituted the static phase culture. After the absorbance at 520 nm of this culture reached a value of approximately 0.5 (5 x 108 CFU/ml, previously determined to be near the mid-log portion of the growth curve), a new experiment was initiated by transferring 1 ml of this culture to 9 ml offresh nutrient broth. This dilution was the log phase culture. The growth of these cultures was followed by measuring the absorbance at 520 nm at various times, using a spectrophotometer (Spectronic 20; Bausch & Lomb, Inc., Rochester, N.Y.). All cultures on which absorbance readings were obtained were prepared in 50-ml nepheloculture flasks (Bellco Glass, Inc., Vineland, N.J.). OSCN- was generated in the test cultures by adding LP (final concentration in medium, 1.0 ,ug/ml), SCN(5.9 mM), and H202 (2.5 mM) to fresh culture medium before addition of bacteria. The preparations were incubated for 5 min at 37°C in a water bath shaker before addition of bacteria. (These were the same conditions which were used to obtain the data in Table 2.) At the end of the incubation, a sample was taken from the medium for OSCN- assay, and the bacteria were added to the medium. Viable counts were obtained by placing an appropriate dilution of the culture being tested on nutrient broth plates containing 1.5% agar (Difco). These samples were collected after the bacteria had been incubated in OSCN--containing medium for 20 min. The controls, containing SCN- alone, LP alone, LP and SCN-, or none of the components of the LP system, did not differ significantly from each other. H202 alone had some effect, which is indicated below. Inhibition of bacterial acid production by the LP system. Bacteria were grown from a frozen culture in a shaker under aerobic conditions at 37°C in nutrient broth. After the cultures had reached stationary phase (>8 x 108 CFU/ml), they were diluted to 108 CFU/ml and allowed to grow to a concentration of 5 x 10' CFU/ml. The bacterial suspension was centrifuged (800 x g), washed with saline, centrifuged, and resuspended in saline. This suspension was used for acid production experiments. In the test experiments, portions (100 or 200 ,ul each) of bacterial suspension were mixed with equal volumes of solutions containing OSCN- which was generated by LP, SCN-, and H202 (concentrations are given in the legend to Fig. 4). In the controls, the bacteria were mixed with equal volumes of phosphatebuffered saline, pH 6.5. After 5 min of incubation at 37°C in the titration cells with constant stirring, 2 ml of unbuffered viability-preserving microbiostatic medium was added. (This medium was developed by Moller to preserve microorganisms during transport [16]. We have found that when used as medium for titration experiments it gives higher and more consistent glucose-stimulated acid production values than does

VOL. 39, 1983

PEROXIDASE SYSTEM EFFECTS ON SALMONELLA

10

TA1535

TA150

HIsG%6 290

p1H6.0 7.5 p 5.7 6.9 7.7 MMIM_I P

PH

P

P1

5.4

250

ZO210 KILLING

230 360

100

FIG. 1. Bactericidal effect (percent killing compared with control) of the LP system on three strains of S. typhimurium. OSCN- (open bars) was generated with LP (final calculated concentration, 2.5 ,ug/ml), SCN- (10 mM), and H202 (700 ,uM) in the presence of bacteria. The OSCN- assays (concentrations indicated in the figure) were made 3 to 10 min after the last addition of H202. The pH of the medium (50 ml) was adjusted by addition of HCI before generation of OSCN-. H202 alone (final calculated concentration, 700 ,uM) (shaded bars) was added as a single portion (0.5 ml).

phosphate-buffered saline. The medium does not interact synergistically with the LP system.) After this addition, pH was controlled with a pH stat titrator (type TTT2) combined with an SBR2 Titragraph and an ABU1 Autoburette (Radiometer, Cleveland, Ohio). The mixture was maintained at pH 6.5 by automatic titration of bacterial acid with 1.0 mM NaOH. Acid production was stimulated by the addition of 100 ,ul of glucose solution to give a final concentration of 1%. The rate of acid production was measured as the slope (AV/At) of the curve of volume of added base (required to maintain the pH at 6.5) versus time taken from the recorder tracing. The scope was converted into nanomoles of acid produced by multiplying AV/At by the concentration of the NaOH solution. The acid production is expressed as nanomoles x minute-' x milliliter-'. Carbonate-free NaOH was prepared fresh daily as previously described (18). OSCN- assay. OSCN- was assayed by reaction with the colored anionic monomer (Nbs) of Nbs2, as described previously by Aune and Thomas (3). The reaction mixture was 64 ,uM in Nbs2 and 60 ,LM in 2mercaptoethanol in 2.0 ml of 0.1 M Tris-hydrochloride buffer (pH 8.0) and contained 50 Fig of catalase per ml. The catalase was added to destroy possible residual H202 which would interfere with the assay. RESULTS Effects of pH on LP system antimicrobial activity. The antimicrobial action of the LP system against the three strains of Salmonella is pre-

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sented in Fig. 1. With hisG46 and TA1530, the percent killing, after 1 h of incubation at 37°C in the presence of high OSCN- concentration, was less than 20%o. The percent killing was slightly higher at pH 5.7 to 6.0 than at pH 7.5 to 7.7. However, the LP system was very effective against the strain with the most permeable cell envelope (TA1535). This effect was not dependent on pH. H202 alone (0.7 mM) had only a slight bacterial effect (less than 15% killing). The disappearance of OSCN- from the medium was much faster in the presence of bacteria than in the absence of bacteria. The half-life of OSCN- was greater than 1 h in the absence of bacteria and less than 20 min in the presence of bacteria. The half-life in the presence of bacteria was similar for each of the three strains. In the presence of bacteria, no OSCN- was detectable after 1 h even with the highest initial concentrations tested. Growth inhibition by the LP system. After a short log phase, hisG46 grew exponentially for 5 h in VB salts-glucose broth (Fig. 2). A log plot of the data gave a doubling time of 1.5 h. When 210 ,uM OSCN- was generated in the presence of bacteria and catalase (50 ,ug/ml) was added, the growth was inhibited so that it did not begin until 5 h later. OSCN- (330 F.M) without catalase 2.5 HISGI 0~

~

~

-

2.0~~~~~ + CATF

1.5 A ~~~~~~/

~

d-CAT

1.00

0.5

/

0

.

5-

TIlE (mm) FIG. 2. Effect of the LP system on the growth of S. typhimurium hisG46. The initial OSCN- concentrations were 210 FM (with catalase [+ CAT]) and 330 pLM (without catalase [- CAT]). OSCN- was generated with LP (final calculated concentration, 2.0 ,ug/ml), SCN- (27 mM), and H202 (0.8 mM) in the presence of bacteria. Catalase (50 ,g/ml) was included in one culture to prevent possible toxicity of unreacted H202. Addition of LP and SCN- without H202 (control) did not have any effect on the growth. A6w, Absorbance at 660 nm.

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PURDY ET AL.

INFECT. IMMUN.

2.5

CONTROL

TA15

/

2.0

E~~~~~~~~

560 1.5-

A660

/

1.0

0.5

-0Wr+

0~~

@_-

CAT

,X,~~~~~~~~~~~~'

*A

o~~~~~~-

0

I

0

o°/o-°P -CAT

t

5

10

TItI (Hm )

FIG. 3. Effect of the LP system on the growth of S.

typhimurium rough mutant TA1535. OSCN-

concen-

trations were 300 F.M (with catalase [+ CAT]) and 340 F.M (without catalase [- CAT]). OSCN- was generated as in the legend to Fig. 2. Catalase (50 p.g/ml) was added into one culture to prevent possible toxicity of unreacted H202. The addition of LP and SCN- without H202 (control) had no effect on growth. A6w, Absorbance at 660 nm.

slightly longer delay, which may have been due to the presence of residual traces of H202 or to the higher concentration of OSCNin the reaction mixture. When the bacteria began to grow, they grew at approximately the same rate as the control regardless of the treatments with OSCN-. The doubling time was 1.8 h (OSCN- with catalase) or 1.9 h (OSCN- without catalase). The rfa- strain, TA1535, was seen to exhibit diauxie (Fig. 3). The carbon source for the first phase of diauxic growth was the citric acid that is used as a buffer in the VB salts (37). Owing to the permeability of the cell envelope of strain TA1535, the citric acid was able to traverse the cell envelope and be used as a carbon source by the bacteria. S. typhimurium has been shown to actively transport citrate by an energy-requiring system. However, when this system is not functioning because of repression by glucose, the cell envelope is essentially impermeable to citrate (9). The biphasic growth was not observed in strain hisG46 because the intact cell envelope prevented the penetration of citrate, nor was diauxie observed in strain TA1535 when citrate was omitted. With strain TA1535, growth after the initial lag phase was slow, reaching the first plateau in 3 to 4 h (Fig. 3). Exponential growth started after 7 to

gave a

9 h with a doubling time of 1.8 h. Again, OSCNcaused a long delay, 8 to 9 h, before growth to the first plateau started. The second plateau (absorbance at 520 nm = 2.1) was reached 12 to 13 h later. With both strains, the presence of LP and SCN- (without H202) had no effect on the growth. H202 alone inhibited the growth of strains hisG46 and TA1535. At H202 concentrations of 0.7 and 1.4 mM, the subsequent growth of strain hisG46 was delayed 1.5 and 3.0 h, respectively. Strain TA1535 was much more sensitive, especially at higher concentrations. Growth of this strain was inhibited for more than 24 h by an initial H202 concentration of 1.4 mM. However, H202 toxicity does not account for the results obtained with mixtures containing SCN- and LP. In these experiments, H202 was always added slowly and in small amounts. Under these conditions, the reaction with SCN- and LP is so rapid that no significant concentration of unreacted H202 is attained. Because strain TA1535 exhibited biphasic growth in the minimal glucose broth but strain TABLE 1. Growth constants for the logarithmic segment of the S. typhimurium hisG46 and TA1535 growth curves for inocula from static and log phase cultures previously incubated in the presence or in the absence of OSCN-a OSCN-b

Strain

Growth phase

hisG46

Static

0

Static

620

(iLM)

Log

0

Log

530

time

(h)

0.8 0.8 0.9 1.0 0.6 0.6 1.0 0.9

Onset of (h) 2.8 2.8 10.6 12.0 2.1 2.1 10.4 10.0

1.2 4.1 1.3 4.1 550 1.1 Static 14.3 1.2 14.5 0 0.9 3.1 Log 0.9 3.1 1.0 13.6 570 Log 1.2 16.7 a The ratio of the absorbance at a given time to the initial absorbance was calculated for each observation. Plots of the natural logarithm (In) of this ratio as a function of time were made for each experiment. The growth constants were calculated from linear least squares analyses of these plots. Doubling time = In 2/ slope; onset of log phase = intercept on the time axis. The minimum number of observations used to calculate the constants presented in this table was 11, with a minimum correlation coefficient of 0.98. b Concentration of OSCN- generated by the LP system just before addition of bacteria.

TA1535

Static

Doubling

0

VOL. 39, 1983

PEROXIDASE SYSTEM EFFECTS ON SALMONELLA

TABLE 2. Effect of growth phase on viability of two strains of S. typhimurium incubated with LP systema Strain

phase

OSCN-b

hisG46

Static Static Log Log

0 620 0 530

Static Static

0 550 0 570

TA1535

% Survival[

100 104 100

84

± ± ± ±

4 3

5 8

100 65 100 0.5

±4 ±6 Log ±4 ± 0.2 Log a LP (1 Fg/ml), SCN- (5.9 mM), and H202 (2.5 mM) (all calculated final concentrations) in phosphate-buffered saline, pH 6.5. b Concentration of OSCN- generated by the LP system just before addition of bacteria. I Mean ± standard deviation. The minimum number of observations used to generate these data was three platings.

hisG46 did not, the experiments were repeated, using nutrient broth with OSCN- generated in the absence of the bacteria to eliminate biphasic growth curves. Subsequent growth curves and viable counts were determined for samples of each strain taken from static and log phase cultures and inoculated into fresh media. The growth curve results are shown in Table 1. In the absence of OSCN-, growth curves for cultures of both strains were similar in shape and maximum observed growth whether they were inoculated from log or static phase cultures. However, as expected, the log phase inocula gave somewhat shorter doubling times and earlier onset of log phase growth than did the static phase inoculations. The incubation of static phase inocula with OSCN- delayed the onset of strain hisG46 log phase growth by 8 h and that of strain TA1535 by more than 10 h. Similar results were obtained by treatment of log phase inocula of these strains with OSCN-. The greater delay in onset of growth for strain TA1535 suggests that increased permeability of the cell envelope results in an increased antibacterial effect of OSCN-. The lower doubling rates seen for the Salmonella strains in nutrient broth as compared with the VB salts-glucose medium are due to the fact that nutrient broth is a more complete growth medium than the minimal glucose medium. Since these experiments cannot distinguish between bactericidal effects (reduction of inoculum size by cell death) and bacteriostatic effects (delay of growth by metabolic inhibition), a viability study was carried out. The static phase of strain hisG46 exhibited 100% viability in the presence of 620 ,uM OSCN-, and the log phase culture of hisG46

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exhibited 84% viability in the presence of 530 ,uM OSCN- (Table 2). Thus, the action of OSCN- toward the static phase culture of strain hisG46 was primarily bacteriostatic, with at least slight bactericidal action toward the log phase culture. When the viability of strain TA1535 was tested, the differences in viability were much greater. The static phase culture of strain TA1535 exhibited 64% viability in the presence of 550 ,uM OSCN-, but the log phase culture exhibited only 0.5% viability in the presence of 570 ,uM OSCN- (Table 2). These experiments again showed that cell envelope permeability was a major determinant in the effectiveness of OSCN- as a bactericidal agent and that growth phase was a secondary determinant. Some lysis of the cells treated with OSCNwas seen, as indicated by the drop in absorbance of suspensions of these two strains after incubation in OSCN-. The degree of lysis was greater in the TA1535 cells than the hisG46 cells and more pronounced for the log phase cultures than for the static phase cultures. Inhibition of bacterial acid production by the LP system. All three strains of S. typhimurium produced acid immediately after addition of glucose. The average rate of apparent hydrogen ion production was 1.8 x 10-16 mol x CFU-' x min-' (range, 0.5 x 10-16 to 4 x 10-16; n = 9), with no obvious differences between different strains. LP (2 ,ug/ml) and SCN- (10 mM) had no effect on acid production. When H202 was also added, OSCN- was generated (100 to 300 ,uM, depending on the amount of H202), and no acid production occurred after glucose addition. As a control, H202 added alone at the concentration used had no effect on acid production. Excess 2-mercaptoethanol (10 mol added per mol of OSCN- in the final reaction mixture) did not reverse the inhibition of acid production. However, when samples of bacteria which were unable to produce acid owing to inhibition by OSCN- were plated on nutrient agar and incubated at 37°C overnight, growth of bacteria was seen, indicating that the inhibition was bacteriostatic rather than bactericidal. The exact nature of the acidic species was not determined. However, Salmonella spp. are able to generate formate, acetate, lactate, and succinate from glucose assimilation, and the evolution of CO2 during the operation of the tricarboxylic acid cycle can give rise to carbonate (17). When OSCN- was generated before bacteria were added (Fig. 4), the critical OSCN- concentration required for complete inhibition of acid production was between 370 and 390 ,uM. DISCUSSION Results from several investigators have confirmed that gram-negative bacteria are less sen-

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INFECT. IMMUN. 300

200 ACID PRODUCTION (NM/MI N/ML)

100

I

-~~~~~ 0

l , O

100

200

1-

-:l EE

300

400

500

OSCN- (PM)

FIG. 4. Inhibition of glucose-stimulated acid production of S. typhimurium hisG46 by the LP system. Acid production (nanomoles x milliliter-' x minute-') is plotted against [OSCN-] (,uM). The incubation mixtures of strain hisG46 all contained 4 x 109 CFU/ml, and various concentrations of OSCN- were generated by LP (final calculated concentration, 4 FLg/ml), SCN- (100 mM), and H202 (30 to 900 ,uM, varied to produce the different concentrations of OSCN-).

sitive to the inhibitory effects of the LP system than are gram-positive bacteria. However, it has been reported that gram-negative organisms, although catalase positive, can be killed by the LP system when exogenous H202 at very low nonbactericidal concentrations is added or is generated enzymatically (6, 13, 22, 25). The system has been shown to be bactericidal for E. coli 0110, 0101 and 9703, Pseudomonas aeruginosa 30/70, Pseudomonas fluorescens EF1998, Klebsiella aerogenes, and S. typhimurium C143/6. However, it was not bactericidal to E. coli ML 308-225 unless the cell envelope was damaged by osmotic shock (34). If SCN- ions were replaced by I- ions in the LP system, killing of intact cells was observed without prior treatment to produce cell envelope damage (34,

35). Reiter and co-workers (6, 13, 22, 25) have reported killing of the intact cells of gramnegative bacteria also by the LP-SCN--H202 combination. As little as 5 ,uM OSCN- was effective against E. coli NCTC 9703 at pH 5.5 (13). At the higher concentrations of LP system components, catalase excreted by E. coli was unable to block antibacterial effects of the LP system (25). Multiplying bacteria (pH 5) were less sensitive to the LP system than nonmultiplying bacteria (pH 3). In the absence of LP, catalase prevented glucose-oxidase-catalyzed accumulation of H202 in the medium. In the presence of LP, catalase had to be added to the medium in very high concentrations to block the bactericidal effects of the LP system. It also was reported (25) that lipopolysaccharides of the cell envelope influence the susceptibility of gram-

negative bacteria to the system and that mutant strains of E. coli and S. typhimurium are killed by the system. However, no details were given for these latter reports. Bjorck et al. (6) used a synthetic LP system with high concentrations of components and found bactericidal effects against E. coli and P. fluorescens as well as a number of gram-negative rods isolated from raw bovine milk. On the other hand, Thomas and Aune (34) found that bactericidal action against the E. coli ML strain with intact cell envelope was observed only at low cell densities or at high H202/SCN- ratios or in incubations of several hours. The relationships between bacteriostatic and bactericidal effects of the LP system are complicated. When SCN- serves as electron donor, the system is bacteriostatic for E. coli at [OSCN-1/cell ratios of 0.5 x 10-16 mol per cell and reaction times less than 30 min (34). However, at 4 x 10-16 mol per cell, bactericidal effects are observed. Despite this concentration effect, the bacteria consume only a small fraction of the available OSCN- (36). Extended incubation (2 h) with excess OSCN- results in nearly complete killing of E. coli, whereas incubation for less than 30 min gives essentially bacteriostatic effects (36). In this case, the bacteria spontaneously recover from the effects of OSCN- 15 to 30 min after the cells are separated from the OSCN-. Our results (Table 1) show that when S. typhimurium is left in the presence of excess OSCN-, recovery times are much longer. Both the hisG46 and TA1535 strains required about 10 h to return to log phase growth after treatment

VOL. 39, 1983

PEROXIDASE SYSTEM EFFECTS ON SALMONELLA

with OSCN-. Similar results were obtained when bacteria from static and log phases were treated with SCN-. The [OSCN-]/cell ratio used in these experiments (Tables 1 and 2) was 1.2 x 10-14 mol/CFU, which is nearly 2 orders of magnitude higher than the ratio for which Thomas and Aune (34) found nearly complete. killing of E. coli. These results indicate that these strains of Salmonella are more resistant to the effects of the LP system than are E. coli. The data in Table 1 do not distinguish between bacteriostatic and bactericidal effects. The data show that in all cases some bacteria survived. However, when the permeability of the cell envelope increased, the cells became more susceptible to killing. For permeable cells, the bactericidal action was clearly more pronounced for cells grown from the log phase than those from the static phase. In view of these findings, it may be possible that all antimicrobial agents which alter the structure and permeability of the bacterial cell envelope (e.g., lysozyme, immunoglobulins) may enhance the antimicrobial action of the LP system. Indeed, it has been shown that secretory immunoglobulin A significantly enhances the antimicrobial action of the LP system against a strain of Streptococcus mutans (31). There are important differences in the experimental conditions under which the data in Tables 1 and 2 were collected. In both sets of experiments, OSCN- was generated in growth medium in the absence of the bacteria. For the growth curve data (Table 1), the OSCN-bacteria mixture was allowed to incubate without further manipulation except for sampling. For the viability experiments (Table 2), samples were diluted and plated after a 20-min incubation with OSCN-. Thus, the data in Table 1 were generated by bacteria recovering from OSCNinhibition while suspended in liquid growth medium, whereas for Table 2, the data came from bacteria recovering on an agar surface. Because of these differences, the data in Tables 1 and 2 are not strictly comparable. The inhibition of acid production has been frequently used as a reliable indicator of the antimicrobial action of the LP system on streptococci (18, 30, 31). During the course of the present work, we found that S. typhimurium also responded to the addition of glucose with an apparent acid production. Although for S. typhimurium the metabolic significance of this acid production is not clear, it was inhibited by the LP system. In these experiments, the acid production of S. typhimurium was much more resistant to inhibition than that of two tested strains of streptococci. The concentration of OSCN- needed for complete inhibition of S. typhimurium hisG46 was as high as 370 to 390 ,uM, whereas the range with S. mutans 10449 is

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60 to 90 ,M and with S. salivarius only 30 to 40 p.M (unpublished data). The acid production of all three strains of S. typhimurium was inhibited by high (>350 ,uM) concentrations of OSCN-, and this inhibition was not reversed by the addition of excess reducing agent (2-mercaptoethanol). When added to untreated suspensions of the bacteria, 2mercaptoethanol has no effect on glucose-stimulated acid production. The killing of E. coli by the myeloperoxidaseCl--H202 system has been shown to proceed via an increased permeability of the cell envelope, and a correlation between permeability increase and killing of bacteria was observed (28). On the other hand, Rest and Spitznagel (26) did not find any significant differences in the killing of the smooth Enterobacteria spp. (including S. typhimurium) and their respective deep rough mutants by the myeloperoxidase-Cl--H202 system. However, these authors concluded that if the peptidoglycan is the preferred substrate for the myeloperoxidase system, as has been suggested (27), then this system might be forming a lowmolecular-weight bactericidal component(s) that easily permeates the outer membrane of the smooth bacteria. Modrzakowski et al. (14) have demonstrated in leukemic polymorphonuclear leukocytes a potent bactericidal fraction of low protein content that showed selective bactericidal activity against a series of rough lipopolysaccharide mutants of S. typhimurium LT2. Susceptibility of the mutants to bactericidal action increased as sugar residues decreased in the lipopolysaccharide components. Thomas (32, 34-36) has studied extensively the mechanism of inhibition of E. coli by the peroxidase-H202-halide (or SCN-) system. With SCN- as a donor, a small part of the OSCN- was consumed rapidly in the oxidation of bacterial sulfhydryls to sulfenyl thiocyanate and sulfenic acid derivatives (34). Oxidation of sulfhydryls to sulfenyl derivatives resulted in an immediate reversible inhibition of respiration, indicating that those components of the inner membrane that are essential to respiration of gram-negative bacteria were also oxidized. The slow bactericidal action was attributed to the further oxidation of sulfenyl derivatives by OSCN- (36). The time course of the disappearance of OSCN-, and of sulfenyl derivatives, was similar to that shown for OSCN- in our experiments with Salmonella sp. in the present work. This loss of sulfenyl derivatives is not reversed by sulfhydryl compounds (36), which may explain why acid production in the strains of S. typhimurium which we exposed to OSCNwas not restored by treatment with excess 2mercaptoethanol. Sulfhydryl oxidation may not be the only

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mechanism of inhibition (12). Treatment of E. coli by the LP system is known to inhibit energydependent glucose and amino acid transport and to cause the release of K+ from cells (13, 22), suggesting inner membrane damage. Law and John (12) have shown that the LP system prevents E. coli from generating a proton motive force across its inner membrane which results in impaired nutrient uptake. This mechanism of inhibition seemed not to be dependent on the oxidation of sulfhydryl groups. Our results confirm the observation that the antimicrobial action of OSCN- is dependent, in part, on its penetration into the cells. Penetration of OSCN- may be limited by its charge (33, 36), but because OSCN--HOSCN are in acidbase equilibrium with a pK of 5.3 (33), HOSCN may be the actual antimicrobial component at low pH. Because of its uncharged nature and solubility in the organic phase (33), HOSCN probably penetrates bacterial cell envelopes more easily. Indeed, the system is more effective at low pH both against gram-positive (10) and gram-negative (25) bacteria. On the other hand, recent studies have shown that in the presence of excess H202, OSCN- may be further oxidized to yield short-lived intermediates, which are even more potent inhibitors (4, 19, 20, 30). Therefore, the concentration of OSCNmay not be a direct measure of the antimicrobial capacity of the LP system, especially since in several experiments, the whole system has been shown to be more effective than OSCN- alone (29, 30). In conclusion, our results show that intact cells of S. typhimurium have considerable resistance to the bactericidal action of the LP system but are susceptible to its bacteriostatic effect. Both the degree of permeability of the cell envelope and the growth phase of these bacteria are determinants of the effectiveness of the LP system as a bactericidal agent. Bactericidal action is increased by increased cell envelope permeability, and susceptible cells are more easily killed in the log phase than they are in the stationary phase of growth. ACKNOWLEDGMENT This study was supported in part by U.S. Public Health Service International Fellowship IF05 TWO 2903-01. LITERATURE CITED 1. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31:347-364. 2. Aune, T. M., and E. L. Thomas. 1977. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80:209-214. 3. Aune, T. M., and E. L. Thomas. 1978. Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion. Biochemistry 17:1005-1010.

INFECT. IMMUN. 4. Bjorck, L., and 0. Claesson. 1980. Correlation between concentration of hypothiocyanate and antibacterial effect of the lactoperoxidase system against Escherichia coli. J. Dairy Sci. 63:919-922. 5. Bjorck, L., 0. Claesson, and W. Schulthess. 1979. The lactoperoxidase/thiocyanate/hydrogen peroxide system as a temporary preservative for raw milk in developing countries. Milchwissenschaft 34:726-729. 6. Bjorck, L., C.-G. Rosen, V. Marshall, and B. Reiter. 1975. Antibacterial activity of the lactoperoxidase system in milk against pseudomonas and other gram-negative bacteria. Appl. Microbiol. 30:199-204. 7. Ellman, G. L. 1959. Tissue sulThydryl groups. Arch. Biochem. Biophys. 82:70-77. 8. Hoogendoorn, H., J. P. Piessens, W. Scholtes, and L. A. Stoddard. 1977. Hypothiocyanite ion, the inhibitor formed by the system lactoperoxidase-thiocyanate-hydrogen peroxide. Caries Res. 11:77-84. 9. Kay, W. W., and M. Cameron. 1978. Citrate transport in

Salmonella typhimurium. Arch. Biochem. Biophys. 190:270-280. 10. Kersten, H. W., W. R. Moorer, and R. Wever. 1981. Thiocyanate as a cofactor in myeloperoxidase activity

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wards Streptococcus lactis and Escherichia coli. J. Gen. Microbiol. 120:513-516. 14. Modrzakowski, M. C., M. H. Cooney, L. E. Martin, and J. K. Spitznagel. 1979. Bactericidal activity of fractionated granule contents from human polymorphonuclear leukocytes. Infect. Immun. 23:587-591. 15. Moldoveanu, Z., J. Tenovuo, J. Mestecky, and K. M. 16.

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Pruitt. 1982. Human milk peroxidase is derived from milk leukocytes. Biochim. Biophys. Acta 718:103-108. Moller, A. J. R. 1966. Microbiological examination of root canals and periapical tissue of human teeth. Scand. J. Dent. Res. 74:1-380. Pelczar, M. J., R. D. Reid, and E. C. S. Chan. 1977. Bacterial metabolism: energy production, p. 169-189. In Microbiology. McGraw-Hill Book Co., New York. Pruitt, K. M., M. Adamson, and R. Arnold. 1979. Lactoperoxidase binding to streptococci. Infect. Immun. 25:304-309. Pruitt, K. M., and J. Tenovuo. 1982. Kinetics of hypothiocyanite production during peroxidase-catalyzed oxidation of thiocyanate. Biochim. Biophys. Acta 704:204-214. Pruitt, K. M., J. Tenovuo, R. W. Andrews, and T. McKane. 1982. Lactoperoxidase-catalyzed oxidation of thiocyanate: polarographic study of the oxidation products. Biochemistry 21:562-567. Pruitt, K. M., J. Tenovuo, W. Fleming, and M. Adamson. 1982. Limiting factors for the generation of hypothiocyanite ion, an antimicrobial agent, in human saliva. Caries Res. 16:315-323. Reiter, B. 1978. Review of nonspecific antimicrobial factors in colostrum. Ann. Rech. Vet. 9:205-224. Reiter, B. 1981. The contribution of milk to resistance to intestinal infection in the newborn, p. 155-195. In H. P. Lambert and C. B. S. Wood (ed.), Immunological aspects of infection in the fetus and newborn. Academic Press, Inc., New York. Reiter, B., R. J. Fulford, V. M. Marshall, N. Yarrow, M. J. Ducker, and M. Knutsson. 1981. An evaluation of the growth promoting effect of the lactoperoxidase system in newborn calves. Anim. Prod. 32:297-306. Reiter, B., V. Marshall, L. Bjorck, and C.-G. Rosen. 1976. Nonspecific bactericidal activity of the lactoperoxidase-

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thiocyanate-hydrogen peroxide system of milk against Escherichia coli and some gram-negative pathogens. Infect. Immun. 13:800-807. 26. Rest, C. F., and J. K. Spitznagel. 1978. MyeloperoxidaseCl--H202 bactericidal system: effect of bacterial membrane structure and growth conditions. Infect. Immun.

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27. Salvaraj, F. J., B. B. Paul, R. R. Strauss, A. A. Jacobs, and A. J. Sbarra. 1974. Oxidative cleavage and decarboxylation by the MPO-H202-C1 antimicrobial system. Infect. Immun. 9:255-260. 28. Sips, H. J., and M. N. Hamers. 1981. Mechanism of the bactericidal action of myeloperoxidase: increased permeability of the Escherichia coli cell envelope. Infect. Immun. 31:11-16. 29. Tenovuo, J. 1979. Formation of the bacterial inhibitor, hypothiocyanite ion, by cell-bound lactoperoxidase. Caries Res. 13:137-143. 30. Tenovuo, J., B. Mansson-Rahemtulla, K. M. Prltt, and R. Arnold. 1981. Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system. Infect. Immun. 34:208-214. 31. Tenovuo, J., Z. Moldoveanu, J. Mestecky, K. M. Pruitt, and B. Mansson-RahemtuLla. 1982. Interaction of specific

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