APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

June 1999, p. 2754–2757 Vol. 65, No. 6

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

BACTOX, a Rapid Bioassay That Uses ProtozoaTo Assess the Toxicity of Bacteria

WOLFRAM SCHLIMME,1 MARCELLO MARCHIANI,1 KURT HANSELMANN,2

AND BERNARD JENNI1*

Novartis Pharma AG, CH-4002 Basel,1 and Department of Microbiology, Institute ofPlant Biology, University of Zurich, 8008 Zurich,2 Switzerland

Received 25 September 1998/Accepted 18 March 1999

A new type of toxicity test based on the protozoan Tetrahymena pyriformis has been developed to assess theoverall toxicity of bacterial strains given as prey. This simple and rapid test is able to detect toxicant-producingbacteria, which may present a biohazard. It can also be used for the risk assessment of microbes designed fordeliberate release.

Many toxicity tests to determine acute and chronic effectsare available for the monitoring and risk assessment of newchemicals and xenobiotics or for the evaluation of the toxicityof environmental pollutants. The choice of the best-suited or-ganism for such bioassays is determined by the substances to beassessed and the sensitivity of the organism (5). For example,engineered cell lines are becoming a tool for the screening ofhormone analogues (8), and algae are effective for testing thephytotoxicity of compounds acting on the photosynthetic path-way (16). Environmental pollutants and toxic substances areassessed with various organisms (1, 7, 9, 11, 15, 18), includingprotozoa, predominantly Tetrahymena (2, 7, 13, 14, 21). Usu-ally, the test organisms are exposed to single chemicals or toenvironmental pollutants present in water but not to entirebacteria.

Bacteria are increasingly involved in many biotechnologicalapplications, including the production of bioactive substances,pest control, and plant protection. To provide the toxicologicaldata for these bacteria as required by, e.g., regulatory author-ities (10), the usual toxicity tests are not applicable since theyare assays based on chemicals in solution. No bioassay to assessthe overall toxicity of microorganisms has been described sofar.

We present a new type of semiquantitative bioassay to assessoverall bacterial toxicity based on the protozoan Tetrahymenapyriformis fed with naturally occurring or genetically modifiedbacteria. Deaths among the protozoa are monitored, and thedeath rate is used to assess the toxicity of the bacteria. T. pyri-formis was chosen because it is a well-recognized standard fortoxicity testing (4, 6, 13–15, 17). The purpose of the BACTOXtest is the detection of the overall toxicity of surreptitiousstrains which synthesize toxic secondary metabolites (toxi-cants) and which may constitute a biohazard. Its purpose is notthe detection of specifically targeted toxins, since bacteria mayproduce several toxic metabolites simultaneously (synergies).This type of bioassay is of ecological relevance, since it moni-tors a trophic interaction at the first level of the food web. Thistest that uses protozoa is the first of its kind that can be usedboth for the detection of bacterial toxicants and for the riskassessment of bacterial strains.

Strains and preparation of microorganisms. The strainsused and their sources are listed in Table 1. Besides the ref-erence strains from official culture collections (American TypeCulture Collection [ATCC], Rockville, Md.; National Collec-tion of Type Cultures [NCTC], London, United Kingdom; andDeutsche Sammlung von Mikroorganismen [DSM], Braun-schweig, Germany), a majority of the strains originated fromscreening programs for the isolation of bacteria producingantifungal or insecticidal substances, e.g., Novartis BiosafetyBactox Collection [NBBC], Novartis Pharma AG, Basel, Swit-zerland). The root-colonizing Pseudomonas fluorescens strainCHA0 (19), coded NBBC 267, was used, and so were someof its derivates, which vary in the production of secondarymetabolites. For example, P. fluorescens CHA0-Rif/pME3424(12), coded NBBC 268, contains a plasmid responsible for theoverproduction of the antibiotics 2,4-diacetylphloroglucinoland pyoluteorin. All bacteria were cultured on tryptone soyagar medium at 25°C for 3 days to allow a direct comparison,though the use of other media might influence the productionof bacterial metabolites. One 1-ml loopful of bacteria was re-suspended into 1 ml of sterile tap water, which corresponds toa McFarland value of approximately 5, or to a concentration of108 to 109 cells ml21 (as determined by dilution and plating ontryptone soy agar).

T. pyriformis GL (ATCC 30327) (3) was grown axenically toa density of approximately 105 protozoa ml21 in 1% proteosepeptone medium enriched with 0.1% yeast extract at 25°C intissue culture flasks. The protozoa were then centrifuged at200 3 g for 1 min. The supernatant was removed, and theprotozoa were resuspended in membrane-filtered (0.22-mmpore size) and autoclaved tap water to a final density of 1 3 104

to 3 3 104 cells ml21. The number of cells was measured witha CASY cell counter (Schaerfe System, Reutlingen, Germany)calibrated to a 30 mM NaCl solution. Before the bacteria wereadded, the protozoa were incubated at 25°C for 30 min toadapt them to the lower osmotic pressure of tap water beforethe start of the assay. Filtered, autoclaved tap water was usedthroughout the test because tap water best mimics the osmo-larity of the natural habitats of Tetrahymena and sterilizationeliminates residual chlorine. If a higher level of standardiza-tion is required, however, distilled water amended with lowconcentrations of minerals (“artificial” freshwater) can also beused (20). Peptones from the protozoan growth medium mayreact with bacterial metabolites and decrease their toxicity.Therefore, the test should be performed only in freshwater,

* Corresponding author. Mailing address: Novartis Pharma AG,K-135.P.16, CH-4002 Basel, Switzerland. Phone: 41-61-696.58.01. Fax:41-61-696.16.49. E-mail: bernard.jenni@pharma.novartis.com.

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even if T. pyriformis readily ingests bacteria in the presence ofdissolved nutrients. Weaker or even false-negative responseswere observed when the test was carried out in the protozoangrowth medium.

Standard bioassay. The test was carried out at 25°C in 12-well non-surface-treated polystyrene plates which allowed forconvenient repetitive microscopical examination. Each wellcontained 1 ml of bacterial suspension to which 500 ml of

protozoan suspension was added. The total volume of 1.5 mlwas large enough to prevent adverse effects of evaporationduring the time course of the assay. The final concentrations ofmicroorganisms were 3 3 104 ml21 for T. pyriformis and 108 to109 ml21 for the bacteria. Bacterial ingestion and lysis of pro-tozoa were observed on an inverted microscope with a 125-foldmagnification. The entire surface of each well was monitoredafter 10 min and after 2, 4, and 8 h. Each bacterial strain wasassayed several times in order to verify the reproducibility ofthe results and the reliability of the test. A protozoan suspen-sion without bacteria was used as a control for every plate.Escherichia coli K-12 strain W3110 (DSM 5911) or strainHB101 (DSM 1607) was also routinely used as a negativecontrol. P. fluorescens NBBC 267 and NBBC 268 were chosenas reference strains for weakly and strongly positive controls,respectively.

The BACTOX test as a tool for monitoring bacterial toxicity.Various bacteria were tested, and in some cases a progressivelysis of the protozoa, which was a clear indication of toxicity,occurred. In the case illustrated in Fig. 1, overall lysis required10 min with the progression presented in Fig. 1a to d. Moretime is needed to obtain the same response for less harmfulbacteria. Microscopic observations should be made periodi-cally in order to monitor the evolution of the protozoan pop-ulation, since lysed protozoan bodies are quickly cannibalizedby the survivors and released proteins may inactivate toxicsubstances. Approximately 10% of the protozoan populationdid not ingest bacteria. As a standard operating procedure, wesuggest monitoring after 10 min and after 2, 4, and 6 to 8 h.Subsequent observations are unnecessary, since harmful effectshave never been observed after 8 h. In order to estimate theintensity of the noxious effect of the tested bacteria, the pro-portion of dead protozoa was determined microscopically. Wepropose an easy-to-judge scale, with rankings from 1 to 5 asshown in Fig. 2. A larger scale would not be appropriate to thesensitivity of the test.

TABLE 1. Bacterial strains used in this study

Bacterium Strain(s) Origina

B. cereus ATCC 11145 ATCCNBBC 134–NBBC 142 H. J. Kempf

B. thuringiensis NBBC 143–NBBC 147 H. J. Kempf

E. coli DSM 1607, DSM 5911, DSM 2840 DSMNBBC 172, NBBC 173 J. FreyATCC 8739, ATCC 10536,

NCTC 9001H.-J. Halfmann

L. lactis NBBC 176–NBBC 178,ATCC 11454, ATCC 14365

K. Seeboth

P. fluorescens NBBC 216–NBBC 266 H. J. KempfNBBC 267–NBBC 274 K. HaasNCTC 4755 H.-J. Halfmann

S. aureus NBBC 280–NBBC 282 K. SeebothATCC 6538, ATCC 6538P,

ATCC 13709, NCTC 7447H.-J. Halfmann

Streptomyces spp. NBBC 298–NBBC 335 K. Seeboth

a H. J. Kempf is from Novartis Crop Protection AG, Basel, Switzerland; J.Frey is from the Institut fur Vet.-Bakteriologie, University of Bern, Bern, Swit-zerland; H.-J. Halfmann is from Novartis Pharma AG; K. Seeboth is fromNovartis Animal Health AG, Basel, Switzerland; and D. Haas is from the Labo-ratoire de Biologie Microbienne, Universite de Lausanne, Lausanne, Switzerland.

FIG. 1. Effect of harmful bacteria on T. pyriformis. The lysis of the protozoa as depicted progressively in panels a to d occurs more or less rapidly, depending onthe bacterial strain fed. After only 10 min of incubation with P. fluorescens NBBC 268, the entire population of protozoa was dead (d). Images were produced byNomarski differential interference contrast microscopy (DIC).

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Typical responses observed with different bacteria. Exam-ples of bacteria which cause different levels of effect are pre-sented in Table 2. No detectable effect was observed withbacteria such as E. coli K-12 or B or Lactococcus lactis. At theother end of the scale, P. fluorescens NBBC 268 killed almostall protozoa within 10 min. For such an immediate effect totake place, one has to assume that the active metabolite(s) wasdissolved in the test medium, since the protozoa did not haveenough time to ingest many bacteria. Other bacteria needed afew hours to develop their noxious effect. Toxicity which de-velops slowly may indicate that the toxic substance is less activeor is synthesized in smaller amounts or that it might be liber-ated or activated during the digestion process.

The sensitivity of the test was assessed with a total of 258strains; among them, 106 strains were active (not shown). Theybelonged to gram-positive or gram-negative bacteria of 22 dif-ferent genera. The high proportion of active strains obtained(41%) is an indication that the test is able to detect varioustoxic substances and is thus applicable to diverse bacterialtypes. We assessed the overall toxicity of bacteria to proto-zoa and not the response of protozoa to specific toxins, sincebacteria commonly produce several toxic substances simul-taneously.

Applicable concentrations of protozoa and bacteria. It wasnecessary to assess the optimal concentration range of proto-zoa. Above a concentration of 105 cells ml21, crowding arti-facts may influence the result, and cell densities below 103

ml21 are too low for a convenient observation. Variations be-tween these concentration limits did not alter the outcome ofthe test results. Thus, highly standardized measurements ofprotozoan cell densities are not necessary.

We have investigated the effects of different bacterial con-centrations on the outcome of the test results. An innocuousE. coli strain applied at 10- or 100-fold the standard concen-tration did not produce any toxic effect (Table 2). A false-positive result due to overcrowding at high bacterial densitiescan thus be excluded. Moreover, an error leading to more thana 10-fold concentration of bacteria is not realistic, because the

resulting turbidity would make reading the test too difficult. A10-fold dilution of a moderately toxic strain which develops itsactivity during the digestion cycle (e.g., P. fluorescens NBBC267) did not change the result. This might be due to feeding

FIG. 2. Scale for the quantification of the BACTOX test. The different stages correspond to the following effects on the protozoan population: 1, no effect, i.e., 100%of the protozoa are healthy; 2, weak, only a few protozoa look significantly impaired compared to the control; 3, strong, with about 50% of the protozoa dead; 4, verystrong, with only a few protozoa still living; and 5, maximum, i.e., all protozoa are dead. Images were produced by DIC.

TABLE 2. Effects of bacterial strains taken as references andof various bacterial concentrations on the protozoaa

Bacterium Strain ConcnEffectb after:

10 min 2 h 4 h 8 h

Control 1* 1* 1* 1*

E. coli B DSM 2840 13 1* 1* 1* 1*

E. coli K-12 DSM 5911 13 1* 1* 1* 1*

L. lactis ATCC 11454 13 1* 1* 1* 1*

P. fluorescens NBBC 268 13 4 5 5* 5*

P. fluorescens NBBC 267 13 1 2 2 2

P. fluorescens NBBC 233 13 1 3 4 4

E. coli NCTC 9001 13 1* 1* 1* 2

E. coli ATCC 8739 13 1* 1* 1* 1*103 1* 1* 1* 1*

1003 1* 1* 1* 1*

P. fluorescens NBBC 267 13 1 2 2 20.13 1 2* 2* 2*

P. fluorescens NBBC 268 13 4 5 5* 5*0.13 2* 3 3 2

a The noxious effect on T. pyriformis is ranked for intensity on a scale from 1to 5, as pictured in Fig. 2. The standard concentration (13) of bacteria was 108

to 109 cells ml21. The experiments were repeated at least 10 times for the strainstested with the 13 concentration and three times when strains were tested withother concentrations.

b *, no variation was observed for any repetition of the experiment.

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behavior, since protozoa track their prey actively and concen-trate it into their digestive vacuoles. A 10-fold dilution of thefast-acting strain NBBC 268, however, weakened the toxic ef-fect by two levels on the scale. This result strengthens the hy-pothesis that in this case, the toxic substance was dissolved inthe solution. To obtain the highest sensitivity, the BACTOX testshould be carried out at the optimal bacterial concentration.

Reliability and reproducibility of the test. The levels given inTable 2 are median values obtained from several repetitionsof the experiment. For all innocuous bacterial strains whichshowed a value of 1 even after 8 h of incubation, higher levelsof toxicity were never observed during at least 10 repetitions ofthe experiment. Variations between toxicity levels observed dur-ing repetitions of the experiment were seldom more than onelevel, and they were due to differences both in visual estima-tions and in the time it took for harmful effects to develop.

Results with bacteria belonging to different taxonomic groups.The test was carried out with various types of bacteria in orderto assess its efficiency and to compare the results with theirclassification in biohazard risk groups (RGs). The BACTOX-positive strains were classified in RG1 and RG2 of either theNational Institutes of Health (NIH) or the European (EU)legislation. Some relevant examples are presented in Table 3.

In line with their classification, the K-12 and B strains ofE. coli showed no activity, while four out of five wild-typeisolates did. No active strains of the food-grade L. lactis bac-teria were detected, but all the pathogenic Staphylococcus au-reus strains tested were harmful (Table 3). The strains of thehighly specific insect pathogen Bacillus thuringiensis were in-nocuous for Tetrahymena, and so were the food-poisoning Ba-cillus cereus strains. The latter species is classified in EU RG2,but the tested strains were not of clinical origin. A significantnumber of strains classified in EU RG1 and akin to the bio-technologically important groups P. fluorescens and Streptomy-ces spp. presented a very high activity. The large range ob-tained on the toxicity scale in those groups reveals that theproduction of active metabolites is more strain specific thanspecies specific. The toxic properties of newly isolated strainsmust therefore be assessed prior to growth on a large scale(10), in order to determine the appropriate biosafety level ofphysical containment. Refined risk assessment or additionalsafety measures can then be implemented for BACTOX-positivestrains classified in EU RG1. Negative BACTOX results, how-ever, do not exclude the possibility of harmful effects.

Conclusion. We present a new type of biosafety test basedon the protozoan T. pyriformis, which is not comparable toother toxicity tests. It allows the monitoring of potentially haz-ardous bacteria taken as such instead of the monitoring ofsingle chemicals. The test organism is also naturally bacteri-

otrophic, which makes it more relevant than conventional tox-icity tests for environmental-impact studies involving deliber-ately released bacteria. This assay can be applied in the riskassessment of genetically modified or wild-type bacteria whichmay exhibit toxic properties.

We thank D. Haas, H. J. Halfmann, H. J. Kempf, U. Schnider, andK. Seeboth for kindly providing bacteria; R. Peck for the protozoanstrain; and C. M. Fischer for corrections.

This work was supported by the Swiss National Science Foundation(Priority Programme Biotechnology, grant no. 5002-35145) and byNovartis Pharma AG.

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TABLE 3. Results of the BACTOX test with somerelevant taxonomic groups of bacteriaa

Bacterium RG(NIH/EU)

No. of testedstrains

% of activestrains

Range on thetoxicity scale

E. coli K-12 or B 1 3 0 1E. coli (wild types) 2 5 80 1–2P. fluorescens 1 60 57 1–5L. lactis 1 5 0 1B. cereus 1/2 10 0 1B. thuringiensis 1 5 0 1S. aureus 2 7 100 2–4Streptomyces spp. 1 38 37 1–5

a By definition, EU RG1 contains only nonpathogenic bacteria, while NIHRG1 does not exclude opportunistic pathogens. A broad range on the toxicityscale indicates that different strains gave different responses, thus revealing theheterogeneity of the taxonomic group considered.

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