oxygen tolerance of human intestinal anaerobes1’ · an anaerobic glovebox isolator similar to...

1762 The American Journal of Clinical Nutrition 30: NOVEMBER 1977, pp. 1762-1769. Printed in U.S.A.

Oxygen tolerance of human intestinalanaerobes1’ 2

Rial D. Rolfe, David J. Hentges, James T. Barrett, and Benedict J. Campbell

In the development of techniques for theisolation and identification of anaerobic

bacteria there have been two major con-cerns: the elimination of molecular oxygenfrom the culture environment and the estab-lishment of a negative oxidation-reductionpotential (Eh) in the culture medium. Some

time ago the question arose regarding therelative importance of these two factors inthe multiplication of anaerobic bacteria. Is

oxygen itself inhibitory or does an adverseEh in the medium interfere with the multi-plication of anaerobic bacteria?

Results of early studies were inconclusive.Some of the data indicated that oxygen was

toxic for anaerobic bacteria and other dataindicated that a positive Eh was the growthlimiting factor (1). In many of these studies

atmospheric oxygen was used to increaseEh precluding differentiation of the effects

of the two factors in inhibiting multiplica-tion of the anaerobes.

In more recent studies where the effectsof oxygen were distinguished from the ef-fects of Eh, molecular oxygen and not ad-verse Eh was the critical factor responsible

for growth inhibition (2, 3). In these stud-ies, a positive Eh was maintained by theaddition of potassium ferricyanide to theculture medium and a negative Eh wasmaintained by the addition of dithiothreitol.When the cultures were aerated none ofthe anaerobic organisms multiplied irrespec-tive of the Eh value of the medium. In theabsence of oxygen, on the other hand, theorganisms multiplied even when the Eh was

poised at an Eh from + 370 my to + 500my with potassium ferricyanide. In related

studies, Onderdonk et al. (4) found thatBacteroides fragilis ssp. fragilis maintainedin a chemostat was sensitive to the effectsof dissolved oxygen but not adverse Eh. In

the absence of oxygen, there was no demon-strable change in viable cell counts from

the steady state even when the Eh wasadjusted to + 300 my by the addition ofpotassium ferricyanide. However, when ox-ygen was introduced into the system, the

viable cell count decreased at a rate compa-rable to the theoretical washout rate for astatic bacterial culture. All of these results

indicate that molecular oxygen, rather thanadverse Eh, per Se, is the factor limiting themultiplication of anaerobes.

When anaerobic cultures were aerated,organisms displayed different degrees of tol-erance to oxygen. In comparing three spe-

cies of anaerobic intestinal bacteria, Waldenand Hentges (3) reported that Peptococcus

magnus was the least tolerant. After 2 hraerobic incubation, no viable organismscould be detected. B. fragilis was interme-

diate in tolerance with no viable organismspresent after five hours. Clostridium perfrmn-gens, the most oxygen tolerant, survived up

to ten hours aerobic incubation. Tally et al.(5) also observed variations in oxygen tol-erance among anaerobic bacteria. The 57strains of bacteria examined were isolatedfrom clinical specimens. Blood agar platesinoculated with the organisms were exposedto room air for varying periods of time, upto 72 hr. Survival time varied considerably,even among organisms of the same species.

The objective of the research describedin this paper was to determine, quantita-tively, the effect of atmospheric oxygen on

the survival of a variety of intestinal anaer-obes. Data are presented which show thatanaerobic intestinal bacteria exhibit a broadrange of tolerance to oxygen. Toleranceappears to be related to the proportion of

From the Departments of Microbiology and Bio-

chemistry, University of Missouri School of Medicine,

Columbia, Missouri 65201.

2 This work was supported by Grant ROl Al 12530

from the National Institute of Allergy and InfectiousDiseases.

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OXYGEN TOLERANCE OF HUMAN INTESTINAL ANAEROBES 1763

bacteria in a population capable of survivalin the presence of oxygen.

Materials and methods

Organisms

The bacteria were isolated from human feces and

from clinical specimens. The clinical specimens, pri-

marily from abscesses, were obtained from the Univer-

sity of Missouri Medical Center Pathology Laboratory.

Fecal isolates were obtained from human feces col-

lected in specimen cups and placed immediately in a

Gas Pak jar (BBL) that had been activated at least 2

hr previously. Within 5 mm after collection, the anaer-

obic jar containing the specimen was introduced into

an anaerobic glovebox isolator similar to that described

by Aranki et al. (6). Anaerobic bacteria isolated from

feces or obtained from clinical specimens were identi-

fied on the basis of morphology, antibiotic sensitivity

patterns, volatile fatty acid production, and by cultural

and biochemical tests described by Holdeman and

Moore (7) and Sutter et al. (8). Included in the studies

were strains of B. fragilis ssp. fragilis,3 B. fragilis ssp.

vulgatus, Fusobacterium necrogenes, Bifidobacterium

infantis, Bifidobacterium adolescentis, variety D, and

C. perfringens. Pseudomonas aeruginosa, a clinical

isolate, and Escherichia coli, a fecal isolate, were

identified according to the procedures outlined in Len-

nette et al. (9). For preparation of inocula, all orga-

nisms were cultivated under anaerobic conditions with

the exception of P. aeruginosa.

Apparatus for oxygen tolerance determinations

A system was devised to examine quantitatively the

effects of atmospheric oxygen on the viability of the

bacteria. The test vessel consisted of a 250 ml spinner

flask (BelIco Glass Co., Vineland, N.J.) with openings

for intake and outlet of air and a stoppered opening

near the bottom of the flask for withdrawal of samples.

The apparatus was placed inside an anaerobic glovebox

isolator (6). Air intake and outlet openings were

connected to the outside of the chamber with rubber

tubing. The test vessel was filled with 135 ml of diluent

(7) and connected to a compressed air line. The airwas filtered through two in-line cotton filters and

bubbled through distilled water before entering the

reaction vessel. The control vessel consisted of an

identical spinner flask containing 135 ml of diluent,

except that the air openings were plugged with cotton.

The control vessel was also placed inside the anaerobic

glovebox isolator.

Oxygen tolerance determinations

The control and test vessels were each inoculated

with approximately l07bacteria per milliliter of dilu-

ent. Samples were removed at 0 time and 20 mm, 45

mm, 1, 11/2, 2, 3, 4, 8, 24, 48, and 72 hr to determineviable populations. Ten-fold serial dilutions were pre-

pared from each sample in diluent. One milliliter of

each dilution was flooded on the surface of a predried

BAF plate (10). After the liquid had been absorbed

by the agar, plates were allowed to incubate in the

anaerobic isolator until colonies were clearly distin-

guishable. Colonies were counted with an electronic

colony counter (New Brunswick Scmentmfmc Co., New

Brunswick, N.J.). For each organism examined, this

procedure was repeated twice and the mean values of

results were determined.

Results

The effects of air exposure on the popu-

lations of the organisms are shown in Fig-ures 1 through 7. Figure 1 illustrates results

obtained with a relatively intolerant orga-nism that survived oxygen exposure for a

short period of time. The organism exam-ined was a fecal isolate, B. fragilis ssp.vulgatus. The abscissa indicates the length

of time the isolate was exposed to atmos-pheric oxygen and the ordinate representsthe viable count determination. It is appar-

ent that the organisms in the anaerobiccontrol vessel maintained a steady popula-tion level whereas the population of the

organisms in the aerated test vessel declinedrapidly so that no viable cells were detected

after 1 �/2 hr.The most oxygen tolerant of the anaero-

bic bacteria was the clinical isolate, C. per-

fringens. Results obtained with this orga-nism are illustrated in Figure 2. Underanaerobic conditions there was some de-crease in viability over a 72-hr period.When the organisms were exposed to at-mospheric oxygen, however, an interestingeffect was observed. Initially, the popula-tion declined rapidly but after 4 hr it stabi-

lized and persisted at a level of approxi-mately 102 organisms per milliliter for 72hr, the duration of the experiment. In con-trast to B. fragilis ssp. vulgatus, the C.

perfringens population contained a propor-tion of cells that could tolerate exposure toatmospheric oxygen. When these suspen-sions were centrifuged and stains were pre-

pared no spores could be detected.

Certain relationships became evidentwhen the oxygen tolerance of the variousisolates was compared. In Figure 3 the

response of two species of Bifidobacteriumto the effects of atmospheric oxygen is

shown. Both species, Bifidobacterium ado-

lescentis, variety D, and Bifidobacterium

Since this manuscript was reviewed, the Interna-

tional Nomenclature Committee has agreed to species

status for the various subspecies of Bacteroides fragilis.

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ANAEROBIC

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1 2

TIME (hours) AFTER INOCULATION

FIG. 1. Oxygen tolerance of B. fragilis ssp. vulgatus (fecal isolate). The points connected by the broken linesrepresent results of two individual experiments and the points connected by the solid line represent average

values of the two experiments.

OXYGEN EXPOSURE

4

5

4

ANAEROBIC

3

OXYGEN EXPOSURE

6 12 18 24 30 36 42 48


FIG. 2. Oxygen tolerance of C. perfringens (clinical isolate). The points connected by the broken linesrepresent results of two individual experiments and the points connected by the solid line represent average

values of the two experiments.

1764 ROLFE ET AL.

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B infantis

B. adolescentis

variety D

B infantis

4 8 12 16 20

variety D

.L24 48


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FIG. 3. Oxygen tolerance of B. adolescentis, variety D, and B. infantis.

infantis, were freshly isolated from feces.Under anaerobic conditions there was littledecrease in the population of either orga-nism over a 48-hr period. When aerated,the populations of both organisms de-creased at essentially the same rate and noviable cells could be detected at 48 hr.Similar responses to the effects of oxygenwere also observed when B. fragilis ssp.fragilis and B. fragilis ssp. vulgatus isolated

from feces, were compared. Neither strainsurvived 4-hr oxygen exposure.

The g�ygen tolerance of anaerobic bacte-ria isolated from clinical specimens wascompared with the tolerance of their coun-

terparts isolated from the fecal flora. Figure4 illustrates results obtained with strains ofB. fragilis ssp. fragilis obtained from thesetwo locations. There was little decrease inthe population of either strain over a 48-hrperiod under anaerobic conditions. Whenaerated, no viable cells could be detected inthe suspension of the fecal strain after 4 hrwhereas the clinical isolate survived up to

48 hr. Initially the effect of oxygen on thetwo isolates was similar but the clinicalisolate contained a proportion of cells thatcould tolerate oxygen exposure for a longer

period of time than the fecal isolate. Whenstrains of C. perfringens from feces andfrom clinical specimens were compared sim-ilar results were obtained. The clinical iso-late contained a larger proportion of oxygentolerant cells than the fecal isolate.

The oxygen tolerance of two aerotolerant

organisms, P. aeruginosa and E. coli, wasalso examined. The results of experimentswith P. aeruginosa, considered to be an

obligate aerobe, are illustrated in Figure 5.

The population of this organism did notdecline during the 72-hr test period eitherunder aerobic or anaerobic conditions. Bycontrast, E. coli, a faculatative organism,was affected by aeration. Figure 6 shows

that the E. coli population unlike that of P.

aeruginosa, declined approximately 100-fold after 72-hr exposure to oxygen. It ap-pears that P. aeruginosa has a better defensemechanism against oxygen toxicity than E.

coli.An illustration of the effects of oxygen

on all bacteria examined is presented inFigure 7. Great variation in oxygen toler-ance among the bacteria is apparent. Essen-

tially, four patterns can be detected. Thosebacteria that were the least tolerant to oxy-

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ClinicalANAEROBIC

Fecal

6

5

4

3

Clinical

4 8 12 16 20 24 28 32 36 40 44 48

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FIG. 4. Oxygen tolerance of B. fragilis ss fragilis. Comparison of the clinical and fecal isolates.

I I I I I I I I I I I �

6 12 18 24 30 36 42 48 54 60 66 72


FIG. 5. Oxygen tolerance of P. aeruginosa.

1766 ROLFE ET AL.

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OXYGEN

EXPOSURE

ANAEROBIC

gen and survived less than 4 hr exposureincluded the fecal isolates B. fragilis ssp.fragilis, B. fragilis ssp. vulgatus, and Fuso-bacterium necrogenes. Intermediate in tol-erance was the clinical isolate B. fragiis

ssp.fragilis,and the fecalisolatesB. infantis

and B. adolescentis, variety D. These bac-teria survived between 24 and 48 hr in thepresence of oxygen. Both fecal and clinicalisolates of C. perfringens survived 72-hr

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ANAEROBIC

OXYGEN EXPOSURE

2

54


FIG. 6. Oxygen tolerance of E. coli.

8

6

2

E. coli(fecal)

necrogenes (fecal)

fragilis sap vulgatus (focal)

6 12 18 24

fragilis sap fra9ilis(fecal)

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FIG. 7. Comparison of the oxygen tolerance of all the bacteria examined.

exposure and were therefore the most tol- Discussionerant of the anaerobic organisms examined.Included for comparison is the effect of The results of our studies confirm otheroxygen on the two aerotolerant organisms, reports (3, 4) that anaerobic bacteria vary

P. aeruginosa and E. coli. considerably in their tolerance to oxygen.

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1768 ROLFE ET AL.

We found, however, that within the samegenus and species, the response of popula-

tions to the effects of oxygen was similar,provided the organisms were obtained from

the same source. This may be a reflectionof similar metabolic activities. If the orga-nisms were obtained from different sources,

on the other hand, the response of thepopulations to oxygen differed, even amongmembers of the same species. We were ableto demonstrate, for example, that popula-tions of organisms isolated from clinicalmaterial contained a larger proportion ofoxygen tolerant cells than their counterpartsisolated from feces. Several factors couldaccount for these differences. The same

precautions were not taken to exclude oxy-gen during collection of the clinical speci-

mens as during processing of the fecal iso-lates. Oxygen .tolerant cells could have been

selected for in the process of obtaining theclinical specimens. Or perhaps, the bacteriaisolated from the clinical specimens wereinitially more tolerant to oxygen than the

bacteria present in the feces. To survive intissue with a high oxygen tension, such as

blood, an anaerobe would need to be some-what tolerant to oxygen. Development oftolerance may be an adaptation, permitting

anaerobic bacteria to survive in ecosystemscontaining oxygen.

The degree of oxygen tolerance of anaer-obic bacteria appears to be a function ofthe proportion of organisms in the popula-tion able to survive in the presence of oxy-gen. This finding may simplify determina-tion of the basis of obligate anaerobiosis,although more bacteria need to be exam-ined before definite conclusions can bedrawn. It is important to know, at the

molecular level, why some organisms in apopulation are more oxygen tolerant thanothers. There has been some work done onthe roles of superoxide dismutase and cata-lase in protecting organisms from the detri-mental effects of oxygen (11). Superoxide

is the negatively charged free radical formof oxygen which is an intermediate in thereduction of oxygen. It is extremely activechemically; more active than hydrogen per-

oxide. Superoxide dismutase converts su-peroxide into hydrogen peroxide and oxy-gen and catalase converts hydrogen perox-

ide into oxygen and water. It is possiblethat among anaerobic bacteria the activities

of these enzymes differ. The basis of oxygen

tolerance and anaerobiosis is a fundamentalconcept relating to the existence of anaero-

bic bacteria. An understanding of this con-cept is critical for effective studies of the

role of anaerobic bacteria in health anddisease and the development of better tech-niques for the isolation and identification ofthese organisms.

Summary

An experimental system was devised toexamine quantitatively the effects of atmos-

pheric oxygen on the viability of anaerobicbacteria freshly isolated from human fecesand from clinical specimens. The bacteriawere suspended in a buffered salt solution

through which oxygen was bubbled. Theapparatus was placed in an anaerobic glovebox isolator and samples were removed atvarious time intervals to determine the ef-fects of oxygen on the viability of the bacte-ria. The anaerobic bacteria varied in theirtolerance to atmospheric oxygen. The de-gree of tolerance appeared to be a genusrelated characteristic. A comparison of theoxygen tolerance of the fecal isolates re-

vealed three general patterns. B. fragilisssp. fragilis, B. fragilis ssp. vulgatus, and F.

necrogenes were the least tolerant to oxy-gen. No viable cells could be detected afteroxygen exposure of 4 hr or less. Intermedi-ate in oxygen tolerance were B. adolescen-tis, variety D, and B. infantis. These orga-

nisms survived less than 48 hr in the pres-ence of oxygen. Of the fecal isolates exam-ined, C. perfringens was the most tolerantto oxygen, surviving oxygen exposure for72 hr or more. The degree of oxygen toler-ance of two anaerobic bacteria, B. fragilisssp. fragilis and C. perfringens, isolatedfrom clinical specimens was compared withtheir fecal flora counterparts. In both cases,the clinical isolate contained a larger pro-portion of oxygen tolerant cells than thefecal isolate. The oxygen tolerance of E.coli and P. aeruginosa was also determined.There was some loss in viability of E. coli

after 72-hr exposure to oxygen. There wasno loss in viability of P. aeruginosa, on the

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other hand, under these conditions. These

results demonstrate that there is a broadspectrum of oxygen tolerance among bacte-ria isolated from the intestinal tract. Toler-ance appears to be related to the proportionof the cells in a population that can survivein the presence of oxygen. a

References

1. HENTGES, D. J., AND B. R. MAIER. Theoretical

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2. O’BRIEN, R. W., AND J. G. MORRIS. Oxygen and

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3. WALDEN, W. C., AND D. J. HENTGES. Differential

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1975.4. ONDERDONK, A. B., J. JOHNSON, J. W. MAYHEW

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