The Effect of Nitric Oxide on Bacteria J. L. SHANK, J. H. SILLIKER, AND R. H. HARPER Research Laboratories, Swift and Company, Chicago, Illinois Received for publication October 23, 1961 ABSTRACT SHANK, J. I,. (Swift and Company, Chicago), J. H. SILLIKER, ANDRI). H. HARPER. The effect of nitric oxide on bacteria. Appl. Nicrobiol. 10:185-189. 1962. Nitric oxide, as well as several other oxides of nitrogen, were assayed for their antibacterial action. It is shown that nitric oxide has virtually no effect on bacteria, whereas both NaNO3 and NaNO2 appear to have either neutral or stimulatory effects. It is suggested that the formation of nitrous acid is mainly responsible for the quantitative as well as the qualitative changes that occur in the bacterial flora of cured meat. A pH-dependent "nitrite cycle" is presented to accouint for the production of nitrous acid in cured meat systems. The addition of nitrate or nitrite to fresh meat ulti- mately results not only in pigment changes, but also, along with sodium chloride, brings about quantitative changes as well as qualitative changes in the bacterial population. MIembers of the genera Pseudomonas and Achromobacter are mainly responsible for the spoilage of fresh meat (Stewart, 1932; Haines, 1933; Ayres, Ogilvy, and Stewart, 1954). In cured meats, on the other hand, this predomi- nately gram-negative flora of aerobic saprophytes is re- placed by gram-positive facultative bacteria of the lactic acid group (unpublished data). Since the chemical and bacteriological aspects of curing occur as a function of nitrate or nitrite addition to meat, it is not unreasonable to postulate that the same general mechanism may be in- volved in both cases. In meats, nitrate (NO3-) is converted to nitrite (NO2-) through bacterial reduction. Nitrite, in the slightly acid environment of meat (pH 5.5 to 6.5), exists in equilibrium with nitrous acid (HN02). Nitrous acid, under the pre- vailing reducing conditions of the meat, is reduced to nitric oxide (NO) which reacts with myoglobin to yield the precursor of myochromogen, the stable red pigment of cured meat (Haldane, 1901; Hoaglund, 1914; Urbain and Jensen, 1940; Jensen, 1954). That nitric oxide may also play an important role in the bacteriology of curing thus becomes an inviting hypothesis. Since nitric oxide reacts with the heme pigments of meat, it could react with the heme-containing enzymes of the gram-negative bacteria, thereby poisoning them. This hypothesis could account, a priori, for the overgrowth of gram-positive, catalase-negative bacteria on cured meat. Although Hatton (1881) states that bacteria connected with the spoilage of meat extracts are able to develop in an atmosphere of nitric oxide, Warburg (1927) concluded that nitric oxide is capable of two distinct reactions with the respiratory enzyme of yeast cells. One reaction is "reversible" and does not permanently affect the cell, whereas the other is "irreversible" and kills the cell. Ingram (1939) reported that the oxygen uptake by Bacillus cereus was inhibited by nitrite (at pH 6), thus implying that the cytochrome system of this organism was involved. This hypothesis was denied by Tarr (1941a) whose data show that it is unlikely that nitrite inhibits growth by affecting the aerobic respiratory enzymes of bacteria. Tarr's conclusion is substantiated by the work of Castel- lani and Niven (1955) who were able to inhibit strepto- cocci with nitrite, even though these bacteria have no heme pigments. Since nitrite is most bacteriostatic under acid conditions (Tarr, 1941a, b) and since nitric oxide would certainly be involved in the pathway of nitrite reduction at reduced pH ranges (Corbet, 1934), a series of experiments was con- ducted to study the effect of the various oxides of nitrogen, including pure nitric oxide, on bacteria. MATERIALS AND METHODS Analytical. Nitric oxide' was washed through 40 % sodium hydroxide before use to remove any residual nitrogen dioxide. High purity nitrogen,2 passed through hot copper threads, was used for flushing the system. Where reported, nitrate was determined quantitatively using the ferrous chloride method (AOAC, 1960). Nitrite was deter- mined colorimetrically with modified Griess reagent (AOAC, 1960). Nitric oxide was qualitatively estimated by entrapment of the gas in agar. That this was nitric oxide could be inferred by the immediate formation of a brown gas upon exposure to oxygen (NO2). Bacteriological. Staphylococcus aureus, Streptococcus durans, and Proteus vulgaris were obtained from the American Type Culture Collection, Washington, D. C. Clostridium 3679, species PA-29, was originally obtained from C. F. Schmidt, Continental Can Company, Chicago, Ill. Lactobacillus K7B was obtained from R. H. Deibel of the American Meat Institute Foundation, Chicago, Ill. Pseudomonas fluorescens was isolated from meat in our laboratory and identified according to Bergey's Manual of 1 The Matheson Company, Inc., East Rutherford, N. J. 2 National Cylinder Gas Company, Chicago, Ill. 185 on December 7, 2020 by guest http://aem.asm.org/ Downloaded from
Transcript
The Effect of Nitric Oxide on Bacteria
J. L. SHANK, J. H. SILLIKER, AND R. H. HARPER
Research Laboratories, Swift and Company, Chicago, Illinois
Received for publication October 23, 1961
ABSTRACTSHANK, J. I,. (Swift and Company, Chicago), J. H.
SILLIKER, ANDRI). H. HARPER. The effect of nitric oxide onbacteria. Appl. Nicrobiol. 10:185-189. 1962. Nitric oxide,as well as several other oxides of nitrogen, were assayedfor their antibacterial action. It is shown that nitric oxidehas virtually no effect on bacteria, whereas both NaNO3and NaNO2 appear to have either neutral or stimulatoryeffects. It is suggested that the formation of nitrous acid ismainly responsible for the quantitative as well as thequalitative changes that occur in the bacterial flora ofcured meat. A pH-dependent "nitrite cycle" is presentedto accouint for the production of nitrous acid in cured meatsystems.
The addition of nitrate or nitrite to fresh meat ulti-mately results not only in pigment changes, but also, alongwith sodium chloride, brings about quantitative changes aswell as qualitative changes in the bacterial population.MIembers of the genera Pseudomonas and Achromobacterare mainly responsible for the spoilage of fresh meat(Stewart, 1932; Haines, 1933; Ayres, Ogilvy, and Stewart,1954). In cured meats, on the other hand, this predomi-nately gram-negative flora of aerobic saprophytes is re-placed by gram-positive facultative bacteria of the lacticacid group (unpublished data). Since the chemical andbacteriological aspects of curing occur as a function ofnitrate or nitrite addition to meat, it is not unreasonable topostulate that the same general mechanism may be in-volved in both cases.
In meats, nitrate (NO3-) is converted to nitrite (NO2-)through bacterial reduction. Nitrite, in the slightly acidenvironment of meat (pH 5.5 to 6.5), exists in equilibriumwith nitrous acid (HN02). Nitrous acid, under the pre-vailing reducing conditions of the meat, is reduced tonitric oxide (NO) which reacts with myoglobin to yield theprecursor of myochromogen, the stable red pigment ofcured meat (Haldane, 1901; Hoaglund, 1914; Urbain andJensen, 1940; Jensen, 1954).That nitric oxide may also play an important role in the
bacteriology of curing thus becomes an inviting hypothesis.Since nitric oxide reacts with the heme pigments of meat,it could react with the heme-containing enzymes of thegram-negative bacteria, thereby poisoning them. Thishypothesis could account, a priori, for the overgrowth ofgram-positive, catalase-negative bacteria on cured meat.
Although Hatton (1881) states that bacteria connectedwith the spoilage of meat extracts are able to develop inan atmosphere of nitric oxide, Warburg (1927) concludedthat nitric oxide is capable of two distinct reactions withthe respiratory enzyme of yeast cells. One reaction is"reversible" and does not permanently affect the cell,whereas the other is "irreversible" and kills the cell.Ingram (1939) reported that the oxygen uptake by Bacilluscereus was inhibited by nitrite (at pH 6), thus implyingthat the cytochrome system of this organism was involved.This hypothesis was denied by Tarr (1941a) whose datashow that it is unlikely that nitrite inhibits growth byaffecting the aerobic respiratory enzymes of bacteria.Tarr's conclusion is substantiated by the work of Castel-lani and Niven (1955) who were able to inhibit strepto-cocci with nitrite, even though these bacteria have noheme pigments.
Since nitrite is most bacteriostatic under acid conditions(Tarr, 1941a, b) and since nitric oxide would certainly beinvolved in the pathway of nitrite reduction at reduced pHranges (Corbet, 1934), a series of experiments was con-ducted to study the effect of the various oxides of nitrogen,including pure nitric oxide, on bacteria.
MATERIALS AND METHODS
Analytical. Nitric oxide' was washed through 40%sodium hydroxide before use to remove any residualnitrogen dioxide. High purity nitrogen,2 passed throughhot copper threads, was used for flushing the system. Wherereported, nitrate was determined quantitatively using theferrous chloride method (AOAC, 1960). Nitrite was deter-mined colorimetrically with modified Griess reagent(AOAC, 1960). Nitric oxide was qualitatively estimated byentrapment of the gas in agar. That this was nitric oxidecould be inferred by the immediate formation of a browngas upon exposure to oxygen (NO2).
Bacteriological. Staphylococcus aureus, Streptococcusdurans, and Proteus vulgaris were obtained from theAmerican Type Culture Collection, Washington, D. C.Clostridium 3679, species PA-29, was originally obtainedfrom C. F. Schmidt, Continental Can Company, Chicago,Ill. Lactobacillus K7B was obtained from R. H. Deibel ofthe American Meat Institute Foundation, Chicago, Ill.Pseudomonas fluorescens was isolated from meat in ourlaboratory and identified according to Bergey's Manual of
1 The Matheson Company, Inc., East Rutherford, N. J.2 National Cylinder Gas Company, Chicago, Ill.
Determinative Bacteriology (Breed, Murray, and Smith,1957).
Lactobacillus K7B was maintained by daily transfers inTrypticase soy broth,3 whereas the other bacteria weresubcultured in brain heart infusion broth.4 Clostridium3679 was enumerated by making serial dilutions in Pep-tone colloid4 followed by 7 days of incubation at 37 C;development of turbidity constituted a positive reaction.S. durans was enumerated using Trypticase soy broth,with 24 hr of incubation at 37 C. The other bacteria wereestimated by plate count procedures on Tryptone glucoseyeast extract agar (TGY).4 For analysis of mixed cultures,P. fluorescens was determined on King's medium (Silliker,Shank and Andrews, 1958); S. aureus on tellurite glycine(Zebovitz, Evans, and Niven, 1955), and S. durans byserial dilution in Azide dextrose broth.4
RESULTSEffect of Nitric Oxide and Nitrogen Dioxide
on Various BacteriaThis preliminary experiment was designed to ascertain
the gross effects of NO and NO2 on bacterial cells. Eighteen-hour broth cultures of the test bacteria were centrifuged,washed in distilled water, and resuspended in 0.001 M phos-phate buffer, pH 7.2. The organisms were individuallyabsorbed onto separate 12.7-mm penicillin assay filter discsand the discs placed in a vacuum desiccator. The desiccatorwas evacuated to 29 in. Hg and backfilled with oxygen-free nitrogen gas. This operation was repeated three times.After the fourth vacuumization, nitric oxide was introducedto atmospheric pressure. The bacteria on the discs werekept in this atmosphere for 30 min. The desiccator wasthen evacuated and refilled with nitrogen. This operationwas repeated three times. The discs were then removedfrom the desiccator under flowing nitrogen and transferredto individual tubes of sterile Trypticase soy broth. Afterbreaking up the filter discs with sterile broken glass, analiquot was removed from each tube for bacteriologicalanalysis. This procedure was repeated with 0.5 % (byvolume) and 1.0 % (by volume) air being injected intothe nitric oxide chamber, resulting in the formation ofapproximately 0.18 % and 0.36 % NO2, respectively.The results of this experiment are given in Table 1
where it is clearly shown that none of the test bacteriawere completely inactivated by exposure to the relativelypure nitric oxide. Except for P. fluorescens, the nitric oxidecounts compared quite favorably with the control(vacuum) counts. That this system was contaminated bytrace amounts of oxygen is suggested by the slight dropin pH (6.1 to 5.8). This could account for the inactivationof P. fluorescens in this experiment. As the amount of airincreased, however, a significant reduction in all bacteriaoccurred. It is noted that the Lactobacillus was the leastsensitive of the test organisms.
I Baltimore Biological Laboratory, Inc., Baltimore, Md.
Effect of Nitrous and Nitric Acids on Bacteria
That pH alone was not responsible for the results shownin Table 1 is indicated by experiments in which 1 % aque-ous solutions of sodium nitrite and sodium nitrate werereacted with an equivalent amount of HCl to form nitricand nitrous acids plus sodium chloride. The acid solutionswere prepared individually and used immediately. Forthis test, 1.0 ml from 18-hr broth cultures was added to 10ml of the acid solutions. Three trials were run for eachtest organism. Contact time between the cells and the acidduring these trials averaged 86 sec, after which the cellswere absorbed onto sterile Millipore discs and washed with100 ml of 0.001 M phosphate buffer. The Millipore filterdiscs were subsequently broken up and counts made onTGY agar. It will be noted from Table 2 that the pHof the nitric acid suspensions was lower than the nitrousacid suspensions, but that the greatest antibacterialeffect was associated with the nitrous acid.
Exposure of bacteria to > 200 ppm NO. Since nitric oxidehas a limited solubility in water (at 25 C, 760 mm, only56.3 ppm nitric oxide will be dissolved), it was decided todetermine the effect of materially increasing the amountof nitric oxide available to react with the bacteria. Accord-ingly, two stainless steel bombs were set up, each contain-
ing 1 liter of 0.85 % NaCl, 0.05 %/0 of K2HPO4 and 0.01 %of KH2PO4 at pH 7.4. Both bombs were autoclaved for45 min at 121 C. One was maintained anaerobic bybubbling nitrogen gas through the buffer. The other wascooled under filtered air. When both reached 25 C (roomtemperature), they were inoculated with the test bacteria.Both bombs were then evacuated for 30 min (29 in. Hg).At this time, nitric oxide was introduced into both bombsuntil a gauge pressure of 40 psi was reached. With thispressure, at 25 C, 203.8 ppm nitric oxide are forced intosolution. Samples for bacteriological examination, under anitrogen blanket, were taken initially (just after inocula-tion), immediately after the pressure reached 40 psi, andafter 15 min at 40 psi. The results are given in Table 3.It is evident that the nitric oxide in the "anaerobic" bombfailed to show any significant antibacterial activity. Incontrast, the "aerobic" bomb showed complete inactiva-tion of the bacteria after 15 min.
Effect of NO and NO2 on Clostridium spores. In attempt-ing to achieve maximal nitric oxide purity under experi-mental conditions, it was found that white mineral oil ischemically inert (within the limits of the experiment) tothe presence of the various oxides of nitrogen. Accordingly,a glass cylinder was completely filled with mineral oiland inverted over a mineral oil reservoir. The cylinder
mineral oil was then displaced with washed nitric oxide.Millipore discs, previously inoculated with Clostridium3679 (a mixture of vegetative cells and spores), were intro-duced on the end of a curved stainless steel fork under themineral oil lock and up into the nitric oxide atmospherefor 30 min. The discs were then removed under flowingnitrogen and broken up in sterile water. Serial dilutionsin Peptone colloid were made. This test was repeated inthe presence of 0.45 % (by volume) and 0.91 % (by volume)nitrogen dioxide formed from the injection of air througha rubber seal into the nitric oxide. Millipore filters inocu-
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EXPERIMENT I
2
6.0 5.5 5.0 4.5 4.0pH
FIG. 1. Effect of pH on the activity of nitrite. Experiment 1:Clostridial counts after 4 days of incubation, initial pH 5 count,after pasteurization = 100,000 PAS679 per g. Experiment 2: (lostrid-ial counts after 2 days of incubation, initial pH 5 coutnt, after pasteuri-zation = 100,000 PAS679 per g.
* In brain heart infusion broth containing 0.15% agar and0.06%XG NaNO2. pH adjustments made with lactic acid. Deter-minations were made after 24 hr at 25 C.
lated with Clostridium 3679 were also exposed to thesesystems for 30 min. Duplicate tests were made for eachcondition. The results, summarized in Table 4, establishthat the vegetative bacterial cell, as well as the spore,
are both inactivated in the presence of nitrogen dioxide,but not in the presence of nitric oxide.
Effect of pH on activity of nitrite in meat against spores
of Clostridium 3679. Sodium nitrite, 160 ppm, was addedto 50 lb of ground meat emulsion, previously inoculatedwith spores of Clostridium 3679 to give 3 X 106 spores
per g. The emulsion was then split into five lots and thepH of each lot adjusted with lactic acid from pH 4.0 topH 6.0 at 0.5 pH increments. Control emulsion, similarlyadjusted but lacking nitrite, was included. After appropri-ate mixing, each emulsion was packaged under nitrogenin thermal death time cans and pasteurized at 64 C for:35 min. The cans were then placed in a 37 C incubator.figure 1 shows the results of two separate experiments.The initial counts, immediately after pasteurization, forboth the control and the nitrite emulsions, were com-
parable. In experiment 1 the clostridial counts of thenitrite-treated meat, after the cans had been incubatedfor 4 days, are given. In experiment 2, the results aftera 2-day incubation are given. It is seen that the bac-tericidal effect is greatest in the pH range 4.5 to 5.5. Oneither side of this range, that is, at pH 6 and at pH 4,the antibacterial effect of the added nitrite was far lesssignificant. It had been reported that, at pH 7.5 or above,nitrite favors the growth of bacteria; at pH 5.6 to 5.8,nitrite exerts a very distinct bacteriostatic action; atpH 5.3 and below, nitrite disappears and again no bac-teriostatic action is found (Henry, Joubert, and Goret,1954). As nitrite disappears in an acid environmentthrough the mediation of nitrous acid, nitrate and nitricoxide are formed (Table 5). These materials represent themore stable end products of nitrite destruction at a low pH.
DISCUSSION
According to Taylor, Wignal, and Cowley (1927),nitrous acid in an aqueous solution decomposes in twostages:
2HN02 N203 + H20 (1)
N203 +N0 (2)
In this scheme, the hydration of nitrogen dioxide wouldaccount, ultimately, for the formation of nitrate, whereasthe nitrite, also resulting, would again be available forsubsequent formation of the anhydride, N203:
H202N02, HNO2 + HN03 (3)
The net result of these reactions is the accumulation ofnitrate and nitric oxide.The fate of sodium nitrate or sodium nitrite added to a
meat system is determined not only by the chemistry ofthese compounds but is profoundly affected by the bio-
logical condition of the muscle itself, as well as the numbersand kinds of bacteria that may be present. The develop-ment of the cured meat color, as indeed the accompanyingantibacterial effect, is not a clear-cut phenomenon foreach may be attributed to many particular components.Cured meat color may be developed by NO, N02, as wellas HN02 (unpublished data), and we have shown in thispaper that NO2 and HN02 are both powerful bactericidalagents. In a system as complex as meat, therefore, one isforced to speak in terms of generalities. If one says that NOcures meat it is meant that a reserve reducing capacityand anaerobiocity has increased the tendency for thissystem to produce and accumulate nitric oxide resultingin a more nearly nitric oxide type of cure. If one says thatnitrous acid is bactericidal it is meant that HN02 hasbecome the main component of an equilibrium mixture thatinvolves N02, N02-, N03-, as well as NO. In other words,maximal color development probably occurs when meatreacts with NO, whereas maximal bacteriostasis occurs inthe presence of HNO2.
In reviewing the literature it was found that the U-curves in Fig. 1 have previously been observed by Tarr(1942). He found that Clostridium botulinum spores grewin an anaerobic fish digest broth (pH 4.6) to which 200ppm sodium nitrite had been added. At pH 5.1 no growthoccurred. When these systems were shaken in the presenceof air, no growth took place. An explanation of this maylie in the fact that on the slightly acid to alkaline sideof the U-curve, sodium nitrite occurs principally as a salt.The early work of Lewis and MIoran (1930) showed thatsodium nitrite, at levels as high as 900 ppm (pH 7.0),actually stimulated the growth of Clostridium sporogenes.The inability of sodium nitrite, per se, to inactivate bac-teria (at the levels commonly used in meat curing) hasalso been reported by Bulman and Ayres (1952) andYesair, Bohrer, and Cameron (1944). In the pH range from4.5 to 5.5, nitrite becomes very bactericidal, becausenitrite exists principally as nitrous acid. Below pH 4.5on the more acid side of the U-curve, nitrous acid is rapidlyconverted to nitrate and nitric oxide. In the presence ofoxygen (e.g., Tarr's aerobic broth), however, nitric oxideis oxidized to nitrogen dioxide which, in an aqueous system,forms nitric and nitrous acids. We hav,e presented data toshow that of these two acids, nitrous acid exhibits by farthe greatest antibacterial activity toward both vegetativecells and spores. In the absence of available oxygen, onthe other hand, only nitric oxide and nitrate accumulateand neither of these possess antibacterial characteristics.In fact, it has been shown that nitrate stimulates aerobicsporeformers (Silliker, Greenberg, and Schack, 1958).The data presented in this paper suggest that nitric
oxide is itself relatively inert. With respect to this con-clusion it is of interest to note the review on this subjectby Gray (1959) who states that in a mixture of NO andNO2 gases, the toxicity of the mixture is about propor-tional to the amount of N2 present. If NO and N03
are excluded as important toxiological agents, it seemsreasonable to postulate that the antibacterial activity ofnitrite, except for the possible formation of hydroxylamine(Lindsey and Rhines, 1932) is due principally to the forma-tion of nitrous acid. The dynamics of nitrous acid produc-tion may now be visualized in a cyclic reaction wherenitrite undergoes a concomitant oxidation-reductionreaction resulting in the formation of nitrate, nitric oxide,and nitrogen dioxide. Nitrogen dioxide, reacting withwater, would generate more nitrate and nitrite with thenitrite re-entering the cycle again (equation 4).
(Hi)
(OH-) {,1K
(HNO,) NO2-
NO:(-
At low pH levels (pH 3 to 4), the cycle rapidly forms N03and NO, virtually exhausting its bactericidal capacity.At intermediate pH levels (pH 4.5 to 5.5) the cycle rotatesmore slowly. The presence of HNO2 is prolonged, therebyincreasing its reaction potential. This is the level of maxi-mal bactericidal activity. At higher pH levels (pH 6 to 7),the equilibrium shifts toward NaNO2, the cycle is pre-vented from functioning, and no bactericidal effectsare noted.
Nitrous acid, formed as indicated above or by the oxida-tion of NO, would have two fundamental areas of reac-tion. One would be with the bacterial cell itself (Quasteland Woolridge, 1927; Philpot and Small, 1938) and theother with various constituents of the medium makingthem unavailable for subsequent metabolism (Castellaniand Niven, 1955). Either or both of these reactions couldresult in bactriostasis. The reason for the selection ofgram-positive bacteria over gram-negative bacteria incured meat systems, apart from the effects of hyper-tonicity, may eventually be explained in these terms.
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