APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p. 784-791 Vol. 45, No. 30099-2240/83/030784-08$02.00/0Copyright C 1983, American Society for Microbiology

Production of Superoxide and Hydrogen Peroxide in MediumUsed To Culture Legionella pneumophila: Catalytic

Decomposition by CharcoalP. S. HOFFMAN,'* L. PINE,2 AND S. BELL3

Microbiology Section, Department ofBiology, Memphis State University, Memphis, Tennessee 381521;Bacteriology Branch, Bacterial Diseases Division, Center for Infectious Diseases, Centers for Disease

Control, Atlanta, Georgia 303332; and Department of Microbiology, University of Georgia, Athens, Georgia306023

Received 9 August 1982/Accepted 23 November 1982

The difficulties associated with the growth of Legionella species in commonlaboratory media may be due to the sensitivity of these organisms to low levels ofhydrogen peroxide and superoxide radicals. Exposure of yeast extract (YE) brothto fluorescent light generated superoxide radicals (3 ,M/h) and hydrogen peroxide(16 ,uM/h). Autoclaved YE medium was more prone to photochemical oxidationthan YE medium sterilized by filtration. Activated charcoals and, to a lesserextent, graphite, but not starch, prevented photochemical oxidation of YEmedium, decomposed hydrogen peroxide and superoxide radicals, and preventedlight-accelerated autooxidation of cysteine. Also, suspensions of charcoal inphosphate buffer and in charcoal yeast extract medium readily decomposedexogenous peroxide (17 and 23 nmol/ml per min, respectively). Combinations ofbovine superoxide dismutase and catalase also decreased the rate of photooxida-tion of YE medium. Medium protected from light did not accumulate appreciablelevels of hydrogen peroxide, and autoclaved YE medium protected from lightsupported good growth of Legionella micdadei. Various species of Legionella (104cells per ml) exhibited sensitivity to relatively low levels of hydrogen peroxide(26.5 ,uM) in challenge experiments. The level of hydrogen peroxide thataccumulated in YE medium over a period of several hours (>50 ,uM) was inexcess of the level tolerated by Legionella pneumophila, which contained nomeasurable catalase activity. Strains of L. micdadei, Legionella dumoffii, andLegionella bozmanii contained this enzyme, but the presence of catalase did notappear to confer appreciable tolerance to exogenously generated hydrogenperoxide.

The addition of activated charcoal to somebacteriological media substantially enhances theisolation and growth of many bacterial patho-gens (2, 9, 23). Activated charcoal (Norit A andNorit SG)-supplemented yeast extract medium(CYE medium) is widely used for the isolationand culture of Legionella pneumophila and re-lated species (5, 6). L. pneumophila does notgrow in autoclaved yeast extract medium (YEmedium) in the absence of added charcoal; how-ever, if the YE medium is sterilized by filtration,the medium supports good growth from largeinocula (13, 25). Ristroph et al. (25) suggestedthat unknown toxic products may be releasedinto the medium during autoclaving. It is gener-ally assumed that these toxic compounds may befatty acids since charcoal can adsorb and there-by detoxify free fatty acids (2, 28). In thisregard, Pine et al. (22) reported that in a chemi-cally defined medium, oleic acid is toxic for L.

pneumophila unless it is added with starch.However, starch does not substitute for char-coal in autoclaved YE medium, suggesting thatthere are additional mechanisms for the stimula-tory action of charcoal.

Activated charcoals have good adsorptiveproperties and are commonly used for adsorbingpollutants out of water and air (27). Charcoal isalso a weak reducing agent and can participate inoxidation-reduction reactions (27, 29). Most ofthe studies on the mechanism of action of char-coal have centered on adsorptive properties, andlittle is known concerning reactions with freeradicals of oxygen.Work in our laboratories has suggested that L.

pneumophila strain Philadelphia-1 possesses lit-tle or no catalase and therefore might be suscep-tible to damage by low levels of hydrogen perox-ide (12a, 22). Low levels of hydrogen peroxideinhibit growth of a variety of bacteria, including

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PEROXIDE TOXICITY TO LEGIONELLAE 785

Neisseria gonorrhoeae (19), Campylobacterje-juni (11), and Spirillum volutans (20). Autooxi-dation and/or photochemical oxidation of com-plex culture media, solutions of amino acids,and vitamins are known to generate appreciablelevels of hydrogen peroxide (3, 4, 11, 19) andsuperoxide radicals (3, 4, 11, 16). Cysteine,which is required in apparent excess of the levelnecessary for nutrition of legionellae (7, 8, 22,26, 30), is known to form hydrogen peroxideupon autooxidation (3, 4). When present insolutions of amino acids, cysteine acceleratesthe production of peroxide (19). Moreover, pho-tooxidation or free radical-mediated oxidation ofmembrane lipids is known to produce toxic lipidperoxides and hydroperoxides (15, 28). The ideathat hydrogen peroxide may form in the mediumused for isolation and culture of L. pneumophilais further supported by the observation of Wa-terworth (31), who found that of several mediatested for photochemical production of perox-ide, only the Mueller-Hinton medium failed toaccumulate hydrogen peroxide. Mueller-Hintonmedium supplemented with starch supportsgood growth of large inocula of L. pneumophilain the absence of added charcoal (7).Our preliminary studies have also indicated

that addition of peroxide and superoxide radicalscavengers, including enzymes such as catalaseand superoxide dismutase (SOD), stimulategrowth in the absence of charcoal. Since nutri-tional studies have not resolved the difficultiesassociated with the growth of legionellae, wedecided to investigate further the possibility thatthe legionellae are sensitive to low levels ofhydrogen peroxide and superoxide and thatcharcoal may act as a protective agent in culturemedia. In this paper we describe additionalmechanisms for charcoal; these mechanismsmay explain the failure of many laboratory me-dia to support growth of legionellae in the ab-sence of charcoal.

MATERIALS AND METHODSBacterial strains and medium. L. pneumophila

strains Philadelphia-1, Knoxville-1, and Bellingham-1,Legionella micdadei strain TATLOCK, Legionellabozmanii strain WIGA, and Legionella dumoffiistrains LB4 and TEX KL were obtained from theculture collection of the Centers for Disease Controland were maintained on Aces-buffered charcoal yeastextract agar (BCYE agar) (21) at 35°C. YE mediumwas prepared by dissolving 10 g of yeast extract (DifcoLaboratories, Detroit, Mich.) in 1 liter of distilledwater. The pH was adjusted to 7.0 before autoclavingor sterilization by filtration. For sterilization by filtra-tion, the broth was first passed through a Whatman no.1 filter and then through a membrane filter (pore size,0.45 ,um; Gelman Sciences, Inc., Ann Arbor, Mich.).Charcoal (0.15%; Sigma Chemical Co., St. Louis,Mo.) was added to the medium before autoclaving or,

for the medium sterilized by ifitration, was autoclavedas a powder and then added after filtration. Whererequired, solutions of cysteine and ferric pyrophos-phate, prepared as described previously (6), wereadded to the YE medium after autoclaving or steriliza-tion by filtration.

Chemicals. Catalase (beef liver grade III), SOD(bovine erythrocyte), o-dianisidine (ODD), Nitro BlueTetrazolium (NBT), and horseradish peroxidase(HRPD) were purchased from Sigma Chemical Co.Each enzyme was tested for activity; catalase had noappreciable SOD activity, and SOD had no detectablecatalase or peroxidase activity. Stock solutions ofODD (5 mg/ml) and NBT (5 mg/ml) were prepared indistilled water, whereas SOD (1 mg/ml), catalase (2mg/ml), and HRPD (1 mg/ml) were prepared in 50 mMpotassium phosphate buffer (pH 7.0). Where required,all solutions were sterilized by filtration and addedaseptically to YE medium. The following charcoalswere tested: Norit A (American Norit Co., Jackson-ville, Fla.), Norit SG and graphite (Sigma ChemicalCo.), BPL (Calgon, St. Louis, Mo.), and CPG (Pitts-burgh Chemical Co., Pittsburgh, Pa.).

Photochemical production of hydrogen peroide andsuperoide in YE medium. Superoxide radicals weredetected in YE broth (without added cysteine andferric pyrophosphate) exposed to fluorescent light (15W) by using the NBT reduction method (11). In thisprocedure, NBT (0.1 mg/ml) was added to two flasksofYE broth, and to one of the flasks 50 to 75 U of SODwas added. Both flasks were exposed to fluorescentlight at a distance of 10 cm. The light intensity at thisdistance was 1.34 mW per cm2 (determined with anenergy meter). Samples (2 ml) were withdrawn fromthe flasks at different times over a 6-h period, and theamount of NBT reduced was determined spectropho-tometrically at 540 nm (3-mi cuvettes; 1-cm light path)by using a molar extinction coefficient of 9,500 forNBT, as determined from a standard curve. Thedifference in reduction ofNBT between flasks contain-ing SOD and flasks without SOD was used to deter-mine the rate of superoxide radical formation. Photo-chemical production of hydrogen peroxide wasdetermined by the ODD-HRPD assay (11). HRPD (20,ug/ml) and ODD (80 pLg/ml) were added to YE broth.Control flasks were stored in the dark and protectedfrom light. Since superoxide radicals reduce oxidizedODD (16) and thus would cause a low determination ofthe hydrogen peroxide produced, we added SOD tosome flasks ofYE medium. There was no oxidation ofODD in the absence of HRPD. The flasks were alsoexposed to fluorescent light as described above for thesuperoxide radical experiment. Samples were taken atdifferent times, and the amount of oxidized ODD wasdetermined spectrophotometrically at 460 nm by usinga molar extinction coefficient of 5,400 (3-ml cuvettes;1-cm light path). The molar extinction coefficientdetermined in this study was obtained for ODD in YEbroth. We found that the extinction coefficient varieddepending on the pH or the type of buffer or at theextremes of the concentration range, as describedelsewhere (10).

Determination of oxygen consumption. A 1.35-mlwater-jacketed cell equipped with a YSI oxygen elec-trode (Yellow Springs Instrument Co., YellowSprings, Ohio) was used to measure oxygen consump-tion (30°C). Oxygen consumption by YE broth was

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786 HOFFMAN, PINE, AND BELL

first determined in the dark to obtain a steady rate, andthen the YE broth was illuminated with a fluorescentlamp. Traces of oxygen consumption were recordedwith a strip chart recorder or a microcomputer inter-faced with the YSI oxygen monitor. Rates of oxygenconsumption were determined by linear regressionanalysis. Various enzymes, such as SOD and catalase,as well as free radical scavengers such as histidine andmannitol, were added to the chamber to determinetheir effects on the rate ofoxygen consumption. Boiledenzymes, bovine serum albumin, and starch served ascontrols. In addition, various types of activated char-coals, graphite, and the CYE medium prepared asdescribed by Feeley et al. (6) were examined. Exceptwhere indicated, all media used in this study wereprepared 24 h before the assay.We tested the ability of various charcoals to decom-

pose hydrogen peroxide by adding 1.5 mM hydrogenperoxide to the chamber. The contents of the chambercontained charcoal plus YE medium (autoclaved andunautoclaved), charcoal suspended in 50 mM potassi-um phosphate buffer (pH 7.0), or CYE medium. Tosimplify measurement of oxygen evolution, wesparged the samples with argon to remove some of thedissolved oxygen. The starting oxygen concentrationwas between 60 and 80% of air saturation. The rate ofoxygen evolution was corrected to take into accountthat 1 mol of oxygen is generated from decompositionof 2 mol of hydrogen peroxide.Growth studies. YE broth and brucella broth, both

containing cysteine and ferric pyrophosphate, wereprotected from light during preparation, sterilizationby autoclaving, and incubation. Unprotected mediumexposed to room light served as a control. The mediawere inoculated with 0.1 ml of a suspension of L.micdadei washed from a BCYE agar plate per 100 mlof medium, and the flasks were incubated statically at37°C for 24 h. Flasks exhibiting growth were thenshaken at 100 rpm on a rotary shaker.The stimulatory effects of starch and charcoal in

chemically defined liquid medium (CDM) were deter-mined by using the medium of Reeves et al. (24). Thismedium was supplemented with combinations of 1%starch and 1 ,ug of oleic acid per ml or with 0.15%charcoal. A cell suspension of L. pneumophila con-taining approximately 2.7 x 10' CFU, as determinedon BCYE agar, was diluted through the 106 dilution,and 0.1 ml of each dilution was added to duplicateplates of CDM. After incubation at 35°C for 5 days,growth was scored visually.

Sensitivity to peroxide challenge. Cells of Legionellastrains grown in BCYE agar were suspended in 10 mlof 10 mM potassium phosphate buffer (pH 7.0) con-taining 0.85% NaCl (PBS). The optical density at 660nm was adjusted to between 0.05 and 0.11 (18- by 160-mm test tubes), as determined with a Beckman modelB spectrophotometer. Such suspensions were useddirectly or diluted 100-fold in PBS. At zero time, 1-mlsamples were removed from the two suspensions anddiluted (10-1 to 10' dilutions), and 0.1-ml sampleswere plated onto duplicate plates of BCYE agar fordetermination of colony-forming units. Hydrogen per-oxide was then added to the remaining cell suspen-sions (26.5 or 55.5 ,uM) at 25°C, and samples wereremoved and plated as described above. The plateswere incubated at 35°C for 5 days, at which time thecolony-forming units were scored. In one experiment

where the number of colonies was too numerous tocount, the colonies were washed from the plates withPBS, and the optical density of a 10-ml suspension wasdetermined as described above.

Catalase activities. Catalase activity was measuredby the method of Beers and Sizer (1). Various strainsof Legionella grown on BCYE agar for 2 to 3 dayswere washed from the surface with PBS, disrupted bysonication (12a), and centrifuged first at 37,000 x g toremove cell debris and then at 100,000 x g for 90 minto obtain the high-speed supernatant. Protein wasestimated by the method of Lowry et al. (14), usingbovine serum albumin as the standard.

RESULTS

Substantial levels of hydrogen peroxide andsuperoxide radicals were detected in YE medi-um exposed to fluorescent light. The intensity ofthe light was approximately twice that found in awell-illuminated laboratory. As Fig. 1 shows,photochemical oxidation of YE broth (withoutcysteine and ferric pyrophosphate) generatedsuperoxide radicals, as evidenced by the reduc-tion of NBT. SOD inhibited NBT reduction, andthe amount of superoxide produced was 3nmol/ml per h. Since superoxide radicals sponta-neously dismutate to form hydrogen peroxide,we also examined YE medium for accumulationof hydrogen peroxide. Figure 2 shows that hy-drogen peroxide was rapidly generated in YEmedium exposed to light, as evidenced by theoxidation of ODD. The superoxide radicals gen-erated photochemically in the medium also af-fected the estimation of hydrogen peroxide, andwhen SOD was added, a marked increase in therate of ODD oxidation was observed (Fig. 2).The amount of hydrogen peroxide generatedwas 16.6 nmollml per h (16.6 ,uM). In 3 h, theamount of hydrogen peroxide accumulated ap-

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.061105204

Time, hrm

FIG. 1. Photochemical generation of superoxideradicals in YE broth. YE medium was exposed tofluorescent light, and NBT reduction was measuredspectrophotometrically at 540 nm. Symbols: 0, YEmedium + NBT; A, YE medium + NBT + SOD (75U).

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PEROXIDE TOXICITY TO LEGIONELLAE 787

1 2 3 4 5

Time, hr

FIG. 2. Photochemical production of hydrcperoxide in YE broth. Hydrogen peroxide producwas determined with ODD and HRPD in the lightand in the dark (U). SOD was added to YE medcontaining ODD and HRPD in the light (0).

proached 50 ,uM. The hydrogen peroxidestable in YE medium since added hydroperoxide remained at nearly constant levels fh, as determined by the ODD-HRPD assay (enot shown).Photochemical oxidation of YE medium

also be monitored by an oxygen probe, andused this method to test the effects not onllcharcoal, but also of added SOD and catalasethe rate of photochemically initiated oxyconsumption. Boiled catalase and SOD, asas bovine serum albumin and starch, servescontrols. If photochemical oxygen consump'proceeds by the following reaction sequenc

402- + 4H+ -- 2H202 + 202

2H202-* 2H2O + 02

then we would predict that with SOD and clase present, 75% of the oxygen consumed ptochemically should be regenerated. As Tabshows, photochemical oxidation of YE medoccurred at a rate of 5 to 10 nmol of oxyconsumed per min per ml of YE broth.higher rate of superoxide and peroxide fortion observed with the oxygen electrode syscompared with the rates observed with b

reduction or ODD oxidation appeared to beto more efficient illumination of the med(smaller volume and stirred). Addition of Sto YE medium caused a 45% decrease in theof oxygen consumption, suggesting that suloxide radicals were produced photochemicaOur results are consistent with equationwhich predicts 50% recovery of the oxyconsumed in the formation of superoxide. Clase was less effective, as predicated from estion 2. Addition of catalase caused a decreas

oxygen consumption of approximately 28%.When SOD and catalase were added to YEmedium, a decrease in oxygen consumption of68% was achieved, which is within 7% of thetheoretical value of 75%. A portion of the oxy-

gen may have been present as hydroxyl radicalsor other reduced forms since mannitol had aslight effect on the rate of oxygen consumption(less than 2% decrease). Histidine had no effecton oxygen consumption. Boiled SOD and cata-lase, as well as bovine serum albumin andstarch, had no effect on the photochemical oxi-dation of YE medium.

Activated charcoal completely inhibited pho-tochemically initiated oxygen consumption inYE medium (Table 2). All of the charcoals used

;,gen in this study had a similar effect on oxygention consumption. Charcoal in buffer or charcoal(^) freshly prepared in BCYE medium consumedlium oxygen. The rate of oxygen consumption by

charcoal in the dark decreased with time andafter 24 h varied between 0.1 and 0.28 nmol ofoxygen per min per ml. In the photochemical

was studies, graphite was the poorest supplement ingen preventing photochemical oxygen consumptionor 8 (2 nmol of oxygen consumed per min per ml ofdata YE medium). However, with the activated char-

coals only a slight increase in oxygen consump-can tion was observed in the light (0 to 0.3 nmol ofwe oxygen consumed per min per ml of YE medi-

y of um). We could not distinguish between preven-, on tion of photochemical oxidation by charcoal andrgen replenishment of oxygen back into the mediumwell by removal of reduced forms of oxygen. Proba-d as bly both mechanisms were involved. When 1.5tion mM hydrogen peroxide was added to the YEe: broth containing the various types of carbon, all

of the charcoals decomposed hydrogen peroxide(1) at rates between 2 and 9 nmol/ml per min. The(2) graphite was less active in decomposing hydro-

ata- gen peroxide (0.9 to 1 nmol/ml per min). Unsup-,ho-ole 1liumrgenThema-;tem4BTduelium'ODrateper-ally.I 1,igen'ata-qua-;e in

TABLE 1. Photochemical oxidation of YE mediumaYE medium 02 consumption % 02supplement(s) (nmol/ml) regenerated

Unsupplemented 5.2-6.9 0(autoclaved)

Catalase 4.02 28SOD 3.56 45Mannitol 4.6 2Histidine 7.2 0SOD + catalase 2.3 68Boiled catalase 5.53 0Boiled SOD 5.71 0Bovine serum albumin 5.06 0.3Starch 6.5 0

a Autoclaved YE medium was exposed to fluores-cent light, and oxygen consumption and evolutionwere measured with an oxygen electrode.

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TABLE 2. Oxygen consumption in the dark and in the light and H202 decompositiona

Medium 02 consumption (nmol/min)b H202 decompositionDark Light (nmol/min)c

YE medium (filtered) 0.39 1.0 <0.5YE medium (autoclaved) 0.12 5.5 <0.5YE medium (autoclaved) + Norit BPL <0.1 <0.3 5.0YE medium (autoclaved) + Norit A <0.1 <0.3 6.44YE medium (autoclaved) + Norit CPG <0.1 <0.3 4.52YE medium (autoclaved) + Norit SG <0.1 <0.3 5.92YE medium (autoclaved) + Graphite <0.1 0.92 2.29CYE 4.4 <0.1 23.6

a Oxygen consumption was measured with an oxygen electrode.b Oxygen consumed in the light was corrected for the rate measured in the dark.Hydrogen peroxide decomposition was determined by measuring oxygen evolution in media to which 1.5

mM hydrogen peroxide had been added.

plemented YE medium did not decompose hy-drogen peroxide, suggesting that hydrogenperoxide was stable in YE medium.The CYE medium also decomposed hydrogen

peroxide. Despite the presence of cysteine andferric pyrophosphate that might react to formhydrogen peroxide, this medium rapidly decom-posed hydrogen peroxide (23 nmol/ml per min).The high rate of peroxide decomposition byCYE medium appeared to be partly due to theaction of cysteine, which is peroxidized to cyste-ine and water. The ferric pyrophosphate supple-ment appeared to have no effect on the decom-position of hydrogen peroxide. Charcoalprepared in potassium phosphate buffer alsodecomposed hydrogen peroxide (17.6 nmol/mlper min), indicating that components of YEmedium (including cysteine and ferric pyrophos-phate) were not required for the observed activi-ty of the charcoal. In addition, fresh suspensionsof charcoal in YE broth decomposed hydrogenperoxide more rapidly than suspensions storedfor 24 h (20 to 40 versus 10 nmol/ml per min).The rate of hydrogen peroxide decompositionwas also dependent on the concentration ofcharcoal (data not shown). The latter experi-ments with CYE medium and phosphate bufferwere performed with 0.2% charcoal rather thanthe 0.15% charcoal used in the other experi-ments. These results demonstrated that activat-ed charcoals in suspension both decompose hy-drogen peroxide and superoxide radicals anddecrease the accumulation of these compoundsin complex media exposed to light.The addition of charcoal to YE medium also

prevented light-accelerated oxidation of cys-teine. Freshly prepared YE broth containing0.04% cysteine autooxidized in the dark at a rateof 40 nmol of 02 consumed per min. In thepresence of fluorescent light, the rate doubled.When charcoal was included in this medium,cysteine oxidation in the dark decreased slightly(30 nmol/ml per min), but more significantly,

charcoal prevented light-accelerated oxidationof the cysteine.Growth studies. If photochemical oxidation of

autoclaved YE medium supplemented with cys-teine and ferric pyrophosphate renders the medi-um inhibitory for growth, then medium protect-ed from light should not be inhibitory. L.micdadei grew poorly in autoclaved YE medium(from a large inoculum) exposed to room light(growth at 48 h), whereas rapid growth occurredwithin 24 h in YE medium protected from light.The unprotected medium eventually reached asimilar turbidity by 72 h. A more dramaticdifference was observed with brucella broth.Medium exposed to light was completely inhibi-tory, whereas the protected medium supportedgood growth by 48 h. These results suggest thatthe difficulties associated with growing legionel-lae in autoclaved media (without charcoal) maybe due to light-induced formation of hydrogenperoxide.

Since starch-supplemented media reportedlyenhanced the growth of L. pneumophila (6, 13),we examined the effect of starch in CDM thatcontained no fatty acids. CDM supportedgrowth to a 10-4 dilution. However, when starchor starch and oleic acid were tested, growth wasslightly inhibited (10-3 dilution). In contrast,charcoal-supplemented CDM supported growthat a 10-7 dilution. This medium contained nofatty acids. These results are consistent with ourfinding that starch had no effect on photochemi-cal oxidation reactions or on decomposition ofhydrogen peroxide and superoxide radicals.This experiment diminishes the probability thattoxic fatty acids are the predominant factor inpreventing growth in most media.Hydrogen peroxide sensitivity and catalase ac-

tivities. L. pneumophila was acutely sensitive tolow levels of hydrogen peroxide (Table 3). Chal-lenge with 26.5 ,uM hydrogen peroxide rapidlykilled strains of legionellae when low cell num-bers were used (104 CFU). However, at higher

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TABLE 3. Effect of hydrogen peroxide on viability of Legionella spp.

StrainH20 dnOptical Colony counts (x 104) at: Optical density of cellStrain concn20 density ___________ suspension at:aStrain concn

of cell Zeoer(4LM) inoculum Zere 0.5 h I h 2h Zime 0.5 h I h 2 h

L. pneumophila Philadelphia-1 26.5 0.11 x 10-2 48 0 0 0L. pneumophila Knoxville-1 26.5 0.06 x 10-2 51 0.8 0 0L. bozmanii WIGA 26.5 0.10 x 10-2 100 20 0 0L. micdadei TATLOCK 26.5 0.07 x 10-2 610 0.13 0 0

L. pneumophila Philadelphia-1 55.5 0.10 1.65 0 0 0L. bozmanii WIGA 55.5 0.095 7.0 3.2 4.3 5.2L. micdadei TATLOCK 55.5 0.09 0.41 0.33 0.65 0.82

a Growth was determined optically at 660 nm as described in the text. Zero-time cells and cells exposed tohydrogen peroxide were plated onto BCYE medium. After 48 h the resulting colonies were washed from thesurface, and the optical density of each suspension was determined as described in the text. The values given arethe averages of duplicate plates.

cell densities (106 CFU) and at 55.5 ,uM hydro-gen peroxide, L. micdadei and L. bozmaniiremained viable (Table 3). Thus, the range ofsensitivity of these strains to hydrogen peroxidefell within the levels of hydrogen peroxide de-tected in YE broth exposed to light. Moreover,it might be anticipated that individual cells maybe even more sensitive to these concentrations.L. pneumophila appeared to be slightly moresensitive to the effects of low levels of hydrogenperoxide than the other species. These resultsmay be explained in part by the fact that L.pneumophila strains had no detectable catalaseactivity (Table 4), whereas the other species hadmeasurable levels of catalase, with L. micdadeihaving the highest level (61 U/mg of protein).

DISCUSSIONWe recently reported that L. pneumophila

contains considerable SOD (25 to 30 U/mg ofprotein), but has little or no catalase activity(12a). The apparent deficiency in catalase sug-gests that these bacteria might be sensitive tolow levels of hydrogen peroxide. Because nutri-tional studies have failed to explain the difficul-

TABLE 4. Catalase activity of Legionella strains

Protein CatalaseStrain Protein activity

(mg/ml) (U/mg of(gm) protein)

L. pneumophila Philadelphia-1 9.9 +aL. pneumophila Knoxville-1 10.0 +L. pneumophila Bellingham-1 5.1L. bozmanii WIGA 4.8 17.9L. dumoffii LB4 5.0 13.8L. dumoffii TEX KL 1.1 33.2L. micdadei TATLOCK 0.6 61.6

a +, Less than 0.1 U of catalase per mg of protein.

ties associated with the isolation and cultivationof the legionellae, sensitivity to low levels ofhydrogen peroxide and superoxide radicals rep-resented a viable alternative explanation. Nutri-tional studies have revealed that the legionellaerequire no vitamins and only a few amino acidsfor growth (7, 8, 23, 25, 30). Thus, these bacteriashould be potentially capable of growing inrather simple media. However, many simple andcomplex culture media fail to support growth ofLegionella spp. from small inocula unless themedia are supplemented with charcoal. All ofthese media appear to share a common problemthat is relieved by charcoal, but not by starch.Thus, determination of the mechanism of actionof charcoal could greatly aid in the resolution ofproblems associated with growth of Legionellaspecies.Our studies suggest that the difficulties associ-

ated with primary isolation of Legionella fromclinical samples, as well as environmental sam-ples, may be explained as follows: (i) legionellaeare sensitive to relatively low levels of hydrogenperoxide and superoxide; (ii) these agents arerapidly generated in media exposed to light byphotochemical oxidation reactions; (iii) activat-ed charcoals enhance growth by preventing pho-tochemical oxidation reactions and by detoxify-ing media of reduced forms of oxygen; and (iv)charcoal also prevents free radical-acceleratedoxidation of cysteine, an essential growth factor.Exposure of YE medium to room light (particu-larly after autoclaving) initiates photochemicaloxidation reactions that lead to accumulation oftoxic levels of hydrogen peroxide. In addition,we found that the level of peroxide generated inYE medium exposed to light (50 ,uM in 3 h) wasin excess of the level tolerated by suspensions oflegionellae (26.5 ,uM). It is reasonable to assumethat with fewer cells (<104 cells), as might bepresent in clinical specimens, even lower con-

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790 HOFFMAN, PINE, AND BELL

centrations of hydrogen peroxide would be in-hibitory. In contrast to the legionellae, Esche-richia coli reportedly can tolerate millimolarlevels of hydrogen peroxide (32).The results of this study also demonstrate that

activated charcoals prevent photochemical oxi-dation reactions in complex media and decom-pose hydrogen peroxide and superoxide radi-cals. Activated charcoals are widely used forpurification of water and air, and the sorptiveproperties of these preparations with regard todetoxification of free fatty acids have been wellstudied (2, 9, 17, 23). However, we have foundlittle information on detoxification of peroxideand various free radicals of oxygen by activatedcharcoals. Charcoals may prevent photochemi-cal oxidation reactions by adsorbing the reactantcompounds, by decomposing reduced forms ofoxygen, or by acting as a trap for free radicals ingeneral. Although we could demonstrate detoxi-fication of hydrogen peroxide by charcoal, noattempt was made to study the other possibili-ties. Activated charcoals are commonly used toadsorb free fatty acids (2, 17, 23), but adsorptionmay not be a major mechanism for permittinggrowth of Legionella. Starch, which also ad-sorbs free fatty acids (22, 28), is only slightlyeffective as a replacement for charcoal. Johnsonet al. (13) reported that starch-supplemented YEmedium sterilized by filtration (but not by auto-claving) supported good growth of variousstrains of Legionella. However, the beneficialeffects .of this medium may be due to the inclu-sion of peroxide scavengers, such as heme anda-ketoglutarate, instead of the added starch. Inaddition, starch could not replace charcoal inour CDM. These observations appear to dimin-ish fatty acid toxicity as the major problemassociated with growth of the legionellae.

Peroxide toxicity is further implicated since L.micdadei could be successfully grown in auto-claved YE medium protected from light duringpreparation and incubation. The effect was evenmore dramatic in brucella broth, in whichgrowth only occurred in the protected medium.Both media, when protected from light, did notaccumulate appreciable levels of hydrogen per-oxide (11). L. micdadei was found to have thehighest level of catalase (60 U/mg of protein),yet these bacteria did not exhibit an appreciableincrease in tolerance compared with the otherstrains of Legionella in peroxide challenge ex-periments. Since catalase and SOD are locatedin the cytoplasm (12a), the results of this studysuggest that targets for damage by peroxide maybe located external to and including the cyto-plasmic membrane. We further believe that acti-vated charcoal acts outside the cell, since it isunlikely that powdered charcoal passes throughthe cytoplasmic membrane.

The problems associated with an acute sensi-tivity to hydrogen peroxide are not unique to thelegionellae, but have been reported for otherfastidious bacteria, including N. gonorrhoeae(19), C. jejuni (11, 12), S. volutans (20), andTreponema pallidum (18). Interestingly, N. gon-orrhoeae reportedly grows on nutrient agar onlyif it is supplemented with charcoal (9). Theobligate microaerophile C. jejuni could be grownaerobically (21% oxygen, air atmosphere) onbrucella agar medium when the medium waseither protected from light or supplemented withSOD or catalase (11). Padgett et al. (20) alsodemonstrated that another microaerophile, S.volutans, could be grown on vitamin-free caseinhydrolysate agar under 12% oxygen when themedium was protected from light. Moreover, ifthe medium was exposed to room light for aslittle as 15 s, the medium was rendered soinhibitory that the bacteria failed to grow evenunder microaerophilic conditions. Preliminarystudies with L. pneumophila indicated that addi-tion of SOD, catalase, or HRPD enhancedgrowth on brucella agar and to a lesser extent onYE medium in the absence of charcoal. Theresults of the enzyme study, as well as studieson the effects of various chemical agents on thegrowth of legionellae, will be presented else-where. Although such results implicate hydro-gen peroxide and superoxide radicals in growthinhibition, our preliminary results with enzymesdo not indicate that they are as efficient or aspractical as the charcoal supplement. The re-sults of our studies on the mechanism of actionor charcoal may have far-reaching implicationsfor a variety offastidious bacteria whose growthis also greatly enhanced on media supplementedwith activated charcoal. Knowledge of the sensi-tivity of legionellae to low levels of hydrogenperoxide and to other reduced forms of oxygenmay be useful for development of improvedprimary isolation media.

ACKNOWLEDGMENTSThis work was supported in part by Public Health Service

grant AI 20139-01 from the National Institutes of Health toP.S.H.

LITERATURE CITED1. Beers, R. F., Jr., and I. W. Sizer. 1952. A spectrophoto-

metric method for measuring the breakdown of hydrogenperoxide by catalase. J. Biol. Chem. 195:133-140.

2. Bey, R. F., and R. C. Johnson. 1978. Protein-free and low-protein media for the cultivation of Leptospira. Infect.Immun. 19:562-569.

3. Carlsson, J., G. P. D. Granberg, G. K. Nyberg, andM.-B. K. Edlund. 1979. Bactericidal effect of cysteineexposed to atmospheric oxygen. Appl. Environ. Microbi-ol. 37:383-390.

4. Carlsson, J., G. Nyberg, and J. Wrethen. 1978. Hydrogenperoxide and superoxide radical formation in anaerobicbroth media exposed to atmospheric oxygen. Appl. Envi-ron. Microbiol. 36:223-229.

APPL. ENVIRON. MICROBIOL.

on May 15, 2018 by guest

http://aem.asm

.org/D

ownloaded from

PEROXIDE TOXICITY TO LEGIONELLAE 791

5. Edelstein, P. H., and S. M. Flnegold. 1979. Use of asemiselective medium to culture Legionella pneumophilafrom contaminated lung specimens. J. Clin. Microbiol.10:141-143.

6. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Lang-ford, J. K. Rasheed, D. C. Mackel, and W. B. Baine. 1979.Charcoal-yeast extract agar: primary isolation medium forLegionella pneumophila. J. Clin. Microbiol. 10:437-441.

7. Feeley, J. C., G. W. German, R. E. Weaver, D. C.Mackel, and H. W. Smith. 1978. Primary isolation mediafor Legionnaires disease bacterium. J. Clin. Microbiol.8:320-325.

8. George, J. R., L. Pine, M. W. Reeves, and W. K. Harrell.1980. Amino acid requirements of Legionella pneumophi-la. J. Clin. Microbiol. 11:286-291.

9. Glass, V., and S. J. Kennett. 1938. The effect of variousforns of particulate carbon on the growth of the gonococ-cus and meningococcus. J. Pathol. Bacteriol. 49:125-133.

10. Guldotti, G., J. P. Colombo, and P. P. Foa. 1961. Enzy-matic determination of glucose: stabilization of colordevelopment by addition of o-dianisidine. Anal. Chem.33:151-153.

11. Hoffman, P. S., H. A. George, N. R. Krieg, and R. M.Smibert. 1979. Studies of the microaerophilic nature ofCampylobacter fetus subsp. jejuni. II. Role of exogenoussuperoxide anions and hydrogen peroxide. Can. J. Micro-biol. 25:8-16.

12. Hoffman, P. S., and T. G. Goodman. 1982. Respiratoryphysiology and energy conservation efficiency of Cam-pylobacterjejuni. J. Bacteriol. 150:319-326.

12a.Hoffman, P. S., and L. Pine. 1982. Respiratory physiologi-cal cytochrome content of Legionella pneumophila. Curr.Microbiol. 7:351-356.

13. Johnson, S. R., W. 0. Schalla, K. H. Wang, and G. H.Perkins. 1982. A simple, transparent medium for study ofLegionella. J. Clin. Microbiol. 15:342-344.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

15. Mead, J. F. 1976. Free radical mechanisms of lipid dam-age and consequences for cellular membranes, p. 51-68.In W. A. Pryor (ed.), Free radicals in biology, vol. 1.Academic Press, Inc., New York.

16. Mlsra, H. P., and I. Fridovich. 1977. Superoxide dismu-tase: a photochemical augmentation assay. Arch. Bio-chem. Biophys. 181:308-312.

17. Mueller, J. H., and P. A. Miller. 1954. Variable factors

influencing the production of tetanus toxin. J. Bacteriol.67:271-277.

18. Norris, S. 1982. In vitro cultivation of Treponema palli-dum: independent confirmation. Infect. Immun. 36:437-439.

19. Norrod, E. P., and S. A. Morse. 1982. Presence of hydro-gen peroxide in media used for cultivation of Neisseriagonorrhoeae. J. Clin. Microbiol. 15:103-108.

20. Padgett, P. J., W. H. Cover, and N. R. Krieg. 1982. Themicroaerophile Spirillum volutans: cultivation on complexliquid media. Appl. Environ. Microbiol. 43:469-477.

21. Pascule, A. W., J. C. Feeley, R. J. Gibson, L. G. Cordes,R. L. Meyerowltz, C. M. Patton, G. W. Gorman, C. L.Carmack, J. W. Ezzell, and J. N. Dowling. 1980. Pitts-burgh pneumonia agent: direct isolation from human lungtissue. J. Infect. Dis. 141:727-732.

22. Pine, L., J. R. George, M. W. Reeves, and W. K. Harrell.1979. Development of a chemically defined liquid mediumfor growth of Legionella pneumophila. J. Clin. Microbiol.9:615-626.

23. Polblok, M. R. 1947. The growth ofH. pertussis on mediawithout blood. Br. J. Exp. Pathol. 28:295-307.

24. Reeves, M. W., L. Pine, S. H. Hutner, J. R. George, andW. K. Harrell. 1981. Metal requirements of Legionellapneumophila. J. Clin. Microbiol. 13:688-695.

25. Ristroph, J. D., K. W. Hedlund, and R. G. Allen. 1980.Liquid medium for growth of Legionella pneumophila. J.Clin. Microbiol. 11:19-21.

26. Ristroph, J. D., K. W. Hedlund, and S. Gawda. 1981.Chemically defined medium for Legionella pneumophilagrowth. J. Clin. Microbiol. 13:115-119.

27. Smisek, M., and S. Cerny (ed.). 1970. Active carbon:manufacture, properties, and applications. Elsevier Pub-lishing Co., New York.

28. Steck, J. L., R. C. Henneberry, and C. D. Cox. 1973.Growth requirements of pathogenic leptospira. Infect.Immun. 7:886-897.

29. Warburg, 0. 1949. Heavy metal prosthetic groups andenzyme action. Oxford Press, London.

30. Warren, W. J., and R. D. Miller. 1979. Growth of Legion-naires disease bacterium (Legionella pneumophila) inchemically defined medium. J. Clin. Microbiol. 10:50-55.

31. Waterworth, P. M. 1969. The action of light on culturemedia. J. Clin. Pathol. 22:273-277.

32. Yoshpe-Purer, Y., and Y. Henls. 1976. Factors affectingcatalase level and sensitivity to hydrogen peroxide inEscherichia coli. Appl. Environ. Microbiol. 32:465-469.

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