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Many toxicity tests to determine acute and chronic effects are available for the monitoring and risk assessment of new chemicals and xenobiotics or for the evaluation of the toxicity of environmental pollutants. The choice of the best-suited organism for such bioassays is determined by the substances to be assessed and the sensitivity of the organism (5). For example, engineered cell lines are becoming a tool for the screening of hormone analogues (8), and algae are effective for testing the phytotoxicity of compounds acting on the photosynthetic pathway (16). Environmental pollutants and toxic substances are assessed with various organisms (1, 7, 9, 11, 15, 18), including protozoa, predominantly Tetrahymena (2, 7, 13, 14, 21). Usually, the test organisms are exposed to single chemicals or to environmental pollutants present in water but not to entire bacteria.

Bacteria are increasingly involved in many biotechnological applications, including the production of bioactive substances, pest control, and plant protection. To provide the toxicological data for these bacteria as required by, e.g., regulatory authorities (10), the usual toxicity tests are not applicable since they are assays based on chemicals in solution. No bioassay to assess the overall toxicity of microorganisms has been described so far.

We present a new type of semiquantitative bioassay to assess overall bacterial toxicity based on the protozoan Tetrahymena pyriformis fed with naturally occurring or genetically modified bacteria. Deaths among the protozoa are monitored, and the death rate is used to assess the toxicity of the bacteria. T. pyriformis was chosen because it is a well-recognized standard for toxicity testing (4, 6, 13-15, 17). The purpose of the BACTOX test is the detection of the overall toxicity of surreptitious strains which synthesize toxic secondary metabolites (toxicants) and which may constitute a biohazard. Its purpose is not the detection of specifically targeted toxins, since bacteria may produce several toxic metabolites simultaneously (synergies). This type of bioassay is of ecological relevance, since it monitors a trophic interaction at the first level of the food web. This test that uses protozoa is the first of its kind that can be used both for the detection of bacterial toxicants and for the risk assessment of bacterial strains.

Strains and preparation of microorganisms. The strains used and their sources are listed in Table 1. Besides the reference strains from official culture collections (American Type Culture Collection [ATCC], Rockville, Md.; National Collection of Type Cultures [NCTC], London, United Kingdom; and Deutsche Sammlung von Mikroorganismen [DSM], Braunschweig, Germany), a majority of the strains originated from screening programs for the isolation of bacteria producing antifungal or insecticidal substances, e.g., Novartis Biosafety Bactox Collection [NBBC], Novartis Pharma AG, Basel, Switzerland). The root-colonizing Pseudomonas fluorescens strain CHA0 (19), coded NBBC 267, was used, and so were some of its derivates, which vary in the production of secondary metabolites. For example, P. fluorescens CHA0-Rif/pME3424 (12), coded NBBC 268, contains a plasmid responsible for the overproduction of the antibiotics 2,4-diacetylphloroglucinol and pyoluteorin. All bacteria were cultured on tryptone soy agar medium at 25°C for 3 days to allow a direct comparison, though the use of other media might influence the production of bacterial metabolites. One 1-µl loopful of bacteria was resuspended into 1 ml of sterile tap water, which corresponds to a McFarland value of approximately 5, or to a concentration of 10⁸ to 10⁹ cells ml¹ (as determined by dilution and plating on tryptone soy agar).

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Standard bioassay. The test was carried out at 25°C in 12-well non-surface-treated polystyrene plates which allowed for convenient repetitive microscopical examination. Each well contained 1 ml of bacterial suspension to which 500 µl of protozoan suspension was added. The total volume of 1.5 ml was large enough to prevent adverse effects of evaporation during the time course of the assay. The final concentrations of microorganisms were 3 × 10⁴ ml¹ for T. pyriformis and 10⁸ to 10⁹ ml¹ for the bacteria. Bacterial ingestion and lysis of protozoa were observed on an inverted microscope with a 125-fold magnification. The entire surface of each well was monitored after 10 min and after 2, 4, and 8 h. Each bacterial strain was assayed several times in order to verify the reproducibility of the results and the reliability of the test. A protozoan suspension without bacteria was used as a control for every plate. Escherichia coli K-12 strain W3110 (DSM 5911) or strain HB101 (DSM 1607) was also routinely used as a negative control. P. fluorescens NBBC 267 and NBBC 268 were chosen as 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 progressive lysis of the protozoa, which was a clear indication of toxicity, occurred. In the case illustrated in Fig. 1, overall lysis required 10 min with the progression presented in Fig. 1a to d. More time is needed to obtain the same response for less harmful bacteria. Microscopic observations should be made periodically in order to monitor the evolution of the protozoan population, since lysed protozoan bodies are quickly cannibalized by the survivors and released proteins may inactivate toxic substances. Approximately 10% of the protozoan population did not ingest bacteria. As a standard operating procedure, we suggest monitoring after 10 min and after 2, 4, and 6 to 8 h. Subsequent observations are unnecessary, since harmful effects have never been observed after 8 h. In order to estimate the intensity of the noxious effect of the tested bacteria, the proportion of dead protozoa was determined microscopically. We propose an easy-to-judge scale, with rankings from 1 to 5 as shown in Fig. 2. A larger scale would not be appropriate to the sensitivity of the test.

View larger version (91K): [in this window] [in a new window]   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 on the 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 by Nomarski differential interference contrast microscopy (DIC).

View larger version (61K): [in this window] [in a new window]   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, very strong, with only a few protozoa still living; and 5, maximum, i.e., all protozoa are dead. Images were produced by DIC.

Typical responses observed with different bacteria. Examples of bacteria which cause different levels of effect are presented in Table 2. No detectable effect was observed with bacteria such as E. coli K-12 or B or Lactococcus lactis. At the other end of the scale, P. fluorescens NBBC 268 killed almost all protozoa within 10 min. For such an immediate effect to take place, one has to assume that the active metabolite(s) was dissolved in the test medium, since the protozoa did not have enough time to ingest many bacteria. Other bacteria needed a few hours to develop their noxious effect. Toxicity which develops slowly may indicate that the toxic substance is less active or is synthesized in smaller amounts or that it might be liberated or activated during the digestion process.

Applicable concentrations of protozoa and bacteria. It was necessary to assess the optimal concentration range of protozoa. Above a concentration of 10⁵ cells ml¹, crowding artifacts may influence the result, and cell densities below 10³ ml¹ are too low for a convenient observation. Variations between these concentration limits did not alter the outcome of the test results. Thus, highly standardized measurements of protozoan cell densities are not necessary.

Reliability and reproducibility of the test. The levels given in Table 2 are median values obtained from several repetitions of the experiment. For all innocuous bacterial strains which showed a value of 1 even after 8 h of incubation, higher levels of toxicity were never observed during at least 10 repetitions of the experiment. Variations between toxicity levels observed during repetitions of the experiment were seldom more than one level, and they were due to differences both in visual estimations 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 order to assess its efficiency and to compare the results with their classification in biohazard risk groups (RGs). The BACTOX-positive strains were classified in RG1 and RG2 of either the National Institutes of Health (NIH) or the European (EU) legislation. Some relevant examples are presented in Table 3.

Conclusion. We present a new type of biosafety test based on the protozoan T. pyriformis, which is not comparable to other toxicity tests. It allows the monitoring of potentially hazardous bacteria taken as such instead of the monitoring of single chemicals. The test organism is also naturally bacteriotrophic, which makes it more relevant than conventional toxicity tests for environmental-impact studies involving deliberately released bacteria. This assay can be applied in the risk assessment of genetically modified or wild-type bacteria which may exhibit toxic properties.