Distribution and diversity of soil protozoa in the McMurdo Dry Valleys of Antarctica

The polar desert soils of the McMurdo Dry Valley region support a limited water film community dominated by flagellates, amoebae, and nematodes. This study describes the protozoa and compares their distribution to nematodes. In 50 samples collected from 12 locations, rotifers and tardigrades were infrequent, and ciliates and testacea were rare. Soil protozoa occurred at all sites but the dominant nematode, Scottnema lindsayae (Timm 1971), did not, indicating soil habitat factors limiting nematode distribution are not limiting to protozoa. In contrast to the nematode species, which are all endemic to Antarctica, there were no endemic protozoan morphospecies found in our samples. The protozoan abundance was several orders of magnitude greater than that of the nematodes, and the species diversity was much greater. Most of the protozoa grew better at lower incubation temperatures. The ubiquitous distribution of protozoa suggests their importance in soil food webs and nutrient cycling in the dry valleys.     

Vertical Micro‐Zonation of Testate Amoebae and Ciliates in Peatland Waters in Relation to Potential Food Resources and Grazing Pressure

The aim of this study was to examine the community structure and vertical micro‐distribution of testate amoebae and ciliates in a raised bog in eastern Poland, as well as to assess the influence of potential food resources (Chl‐a, bacteria, heterotrophic flagellates) and predators (rotifers and copepods) on protozoa communities. Samples were taken from surface, bottom and interstitial waters. At each type of micro‐habitat and each sampling date water was sampled using a plexiglass corer or mini‐piezometers. Additionally, in order to evaluate grazing pressure, field enclosures were used in which metazoan abundance and composition was manipulated by size‐fractionation. Over experiments, metazoan populations shifted from dominance of rotifers to copepods. In the first experiment, with rotifers dominating, metazoa had only a modest predatory impact on the protozoa. In contrast, the second experiment, with copepods prevailing, demonstrated a clear top‐down control of the protozoa communities by metazoan. The density and biomass of protozoa significantly differed between the studied stations, with the lowest numbers in the interstitial water and the highest in the surface water. Surface sampling were dominated by mixotrophic taxa, whereas the deepest sampling level was characterized by increase in the proportion of bacterivore species. These differences between micro‐habitats may be due to differences in environmental conditions (food resources and grazing pressure). Ordination analysis revealed that bacteria can strongly regulate the abundance and taxonomic composition of protozoa in the bottom and interstitial waters. Metazoan predators could be the main regulators of protozoa communities in surface water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Protozoan Bacterivory and Escherichia coli Survival in Drinking Water Distribution Systems

The development of bacterial communities in drinking water distribution systems leads to a food chain which supports the growth of macroorganisms incompatible with water quality requirements and esthetics. Nevertheless, very few studies have examined the microbial communities in drinking water distribution systems and their trophic relationships. This study was done to quantify the microbial communities (especially bacteria and protozoa) and obtain direct and indirect proof of protozoan feeding on bacteria in two distribution networks, one of GAC water (i.e., water filtered on granular activated carbon) and the other of nanofiltered water. The nanofiltered water-supplied network contained no organisms larger than bacteria, either in the water phase (on average, 5 × 10⁷ bacterial cells liter⁻¹) or in the biofilm (on average, 7 × 10⁶ bacterial cells cm⁻²). No protozoa were detected in the whole nanofiltered water-supplied network (water plus biofilm). In contrast, the GAC water-supplied network contained bacteria (on average, 3 × 10⁸ cells liter⁻¹ in water and 4 × 10⁷ cells cm⁻² in biofilm) and protozoa (on average, 10⁵ cells liter⁻¹ in water and 10³ cells cm⁻² in biofilm). The water contained mostly flagellates (93%), ciliates (1.8%), thecamoebae (1.6%), and naked amoebae (1.1%). The biofilm had only ciliates (52%) and thecamoebae (48%). Only the ciliates at the solid-liquid interface of the GAC water-supplied network had a measurable grazing activity in laboratory test (estimated at 2 bacteria per ciliate per h). Protozoan ingestion of bacteria was indirectly shown by adding Escherichia coli to the experimental distribution systems. Unexpectedly, E. coli was lost from the GAC water-supplied network more rapidly than from the nanofiltered water-supplied network, perhaps because of the grazing activity of protozoa in GAC water but not in nanofiltered water. Thus, the GAC water-supplied network contained a functional ecosystem with well-established and structured microbial communities, while the nanofiltered water-supplied system did not. The presence of protozoa in drinking water distribution systems must not be neglected because these populations may regulate the autochthonous and allochthonous bacterial populations.     

The temporal and spatial patterns of protozooplankton abundance in a eutrophic temperate lake

The temporal and spatial distribution of planktonic protozoa of Esthwaite, a eutrophic lake, was investigated at 7–10 day intervals between February to October 1988. Sarcodine protozoa were of little significance, the plankton was dominated by ciliates and flagellates. Ciliate density peaked in late May to early June with densities reaching 9.2 × 10³ 1^-1. There was considerable variation in spatial distribution and greatest species diversity occurred in March/April. After the establishment of summer stratification the planktonic ciliates were confined to water of >25% oxygen saturation in the water column. Oligotrichs, particularly the genus Strombidium and tintinnids, and peritrichs dominated the ciliate assemblages. There was no correlation between chlorophyll a concentrations and ciliate numbers, but a correlation was apparent between ciliate numbers and flagellate density. There were significant differences between the protozooplankton communities at the different sampling sites in the lake.     

拒马河原生动物和底栖动物初步调查及水质分析

2003年3月和4月,对拒马河(北京段)的原生动物及底栖动物群落结构和物种多样性进行了调查分析,并且就其水质现状及水生动物种类和数量变化对环境污染的指示意义加以探讨。共鉴定出原生动物125种,底栖动物17种。结果表明,拒马河水生动物种类较为丰富,物种多样性高,水质生物指标综合评价拒马河水体受到轻度和中度污染。此外,通过对软体动物、环节动物、水生昆虫在不同站点分布的差异分析,表明河流水体污染可导致底栖动物多样性明显降低。从水生生物指示种的变化,可预测水质的变化。     

The horizontal distribution of plankton in a deep, oligotrophic lake—Loch Ness, Scotland

1. The horizontal distribution of plankton in Loch Ness, a deep, oligotrophic lake with a simple trench morphometry, was studied on three occasions in 1993. Samples were collected from ten stations spaced along the length of the loch and the abundance of algae, bacteria, protozoa, rotifers and crustacean zooplankton was determined. 2. Horizontal variability was found to be greatest for the metazoan zooplankton and for the algae, especially two cryptomonad flagellates. Bacteria and heterotrophic protozoa showed relatively little horizontal variability. The degree of horizontal variability was not sufficient seriously to affect studies of the seasonality of plankton abundance conducted from a single sampling station. 3. Gradients of plankton distribution along the length of the loch were dependent on the recent wind history. The direction of the gradient could readily reverse with a shift in wind direction. These results indicate that the horizontal distribution of plankton in Loch Ness is more dependent on wind-induced water circulation patterns than on differential growth of plankton in water masses of differing chemistry.     

Spatio-temporal Patterns of Protozoan Communities in a Meso-eutrophic Reservoir (Esch-sur-Sûre,Luxembourg)

The spatio-temporal distribution of the heterotrophic nanoflagellates (HNF) and ciliates was monitored in the reservoir of Esch-sur-Sûre during the year 1999. Three main periods of protozoan development were observed, in early April, early May, and in July. On the basis of the seasonal dynamics, it appeared that the early spring development of protozoa was probably not controlled by resources or predators. The second protozoan development was progressively controlled by the increase of metazooplankton density that led to the clear water phase characterised by very low protozoan densities and biomasses. A summer development of protozoa was possible thanks to the development of bacteria and moderate metazooplankton densities due to the appearance of non-edible algae. Prorodontida, Halteriida and Strombidiida were the dominant ciliates in the upper part of the water column. A development of Tintinnida was moreover observed in spring whereas Philasterida and Sessilida developed in winter and summer. Ciliates occupied the entire water column in spring and were concentrated in the epilimnion and the metalimnion during the summer period where they fed on bacteria and algae.     

三峡水库运行期间原生动物群落的时空异质性

于2010年10月—2011年6月三峡水库正常运行周期内对库区干流原生动物进行调查,研究其空间分布及水库周期排蓄期间的变化。共检测到原生动物99种,蓄水后纤毛虫有增多的趋势。水库运行的不同阶段优势种不同,大致演变为:砂壳纤毛虫(蓄水期)—非砂壳类纤毛虫(高位运行期间)—有壳肉足虫(低位运行期间)。不同水域优势种也存在差异,从上游到下游特征指示种变化为:有壳肉足虫(变动回水区)—纤毛虫(湖泊区)。结果表明,三峡水库原生动物的分布具有明显的时空异质性(P0.05),影响原生动物时空分布的主要因素有透明度、温度、电导率和叶绿素a。原生动物平均密度为952.19个/L,平均生物量为8.14μg/L。蓄水期上游现存量高于下游,低位运行期间则低于下游。原生动物Marglef和Shannonn-Weiver多样性指数平均值分别为3.78和2.18,1月份最低,6月份最高。蓄水175 m后上游变动回水区原生动物具有较高的丰度。水库冬蓄夏排的运行模式模糊了河流本身的季节变化,使原生动物的种类和现存量更多的受水库水动力学的影响,使水体理化因子和水文特征呈现明显的时空差异,最终形成原生动物种群的时空异质性分布。     

Relative effects of bacterial and protozoan predators on survival of Escherichia coli in estuarine water samples.

The relative effect of protozoan and bacterial predators on the survival of Escherichia coli in estuarine water samples was examined. Predacious protozoa exerted their major influence on E. coli destruction during the first 2 days of a 10-day-decline period. Inhibition of protozoa after day 2 had little effect on E. coli survival. Bacterial predators also contributed to E. coli destruction but in natural estuarine water samples were maintained at lower levels due to "grazing" by predacious protozoa.     

Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella-positive environmental samples

Aquatic protozoa are natural hosts of the human pathogen Legionella pneumophila. The fluorescence labeled 16S rRNA-targeted oligonucleotide probe LEGPNE1 has recently been shown to specifically detect extracellular legionellae as well as intracellular legionellae parasitizing protozoa. In this study we designed oligonucleotide probes which are complementary to distinct regions of the 18S rRNA of the Legionella host organisms of the genera Hartmannella and Naegleria. The specificity of the probes, HART498 and NAEG1088, was tested by in situ hybridization of various laboratory reference strains. In order to evaluate the fluorescent probes for environmental studies three selected Legionella-positive cold water habitats were examined for the presence of these protozoa. Traditional culture methods followed by morphological identification revealed an almost consistent presence of Naegleria spp. in cold water habitats. Other protozoa species including Acanthamoeba spp., Echinamoeba spp., Hartmannella spp., Platyamoeba placida, Saccamoeba spp., Thecamoeba quadrilineata, and Vexillifera spp. were found sporadically. Concomitant analysis of the pH, conductivity and temperature of the water samples revealed no preference of Legionella or the respective protozoa for certain environmental conditions. The specificity of the newly designed 18S rRNA probes demonstrates that they are valuable and rapid tools for the identification of culturable environmental protozoa.     

Predation rates of flagellate and ciliated protozoa on bacterioplankton in a river

Abstract Predation rates of flagellate and ciliate protozoa on the bacterioplankton of Butrón River (Spain) were determined from FLB (fluorescently labelled bacteria) uptake rates. Bacterial and ciliate protozoa counts were higher when higher water temperature was recorded. Flagellate counts did not show this pattern, which suggested predation of flagellates by other organisms, or some other different nutritional mode besides phagotrophy. Average individual ciliate predation rates were up to 40-times higher than those of flagellates. These results were compared with similar data obtained from other authors in several aquatic systems. However, the population predation rates of flagellate protozoa were on average 6-times higher than that of ciliate protozoa, due to the low population numbers of the latter. Thus, flagellate protozoa can be considered as more important bacterial consumers than ciliates in this aquatic system.     

Explanation for the decline of bacteria introduced into lake water

The sizes of the populations of individual bacterial species diminished following their addition to water from lakes with different trophic levels at temperatures of 5, 10, 15, and 30°C. Some species persisted after their initial reduction in cell numbers, but others were undetectable after 3 to 15 days. The decline of these introduced bacteria was not a result of their inoculation at higher densities than are found in nature. The death of most of the test species was not the result of starvation, abiotic factors, bdellovibrios, or bacteriophages. Despite the presence of lytic bacteria, the lake water did not have lytic activity against the test species. Protozoan predation was a significant factor in the fall in bacterial population sizes because protozoa increased in numbers as the bacterial density fell, the suppression of protozoa led to the elimination or delay of the decline of the bacteria, and the addition of protozoa to lake water in which indigenous protozoa were suppressed produced the same pattern of bacterial elimination as in untreated lake water.     

Seasonal change of protozoa and micrometazoa in a small pond with leaf litter supply

Protozoa and micrometazoa were observed in a small pond with leaf litter over a 2 year period. The total number of taxa observed in this pond was 83 protozoans and 30 micrometazoans, and the correspondence of each taxon and the habitats of surface water, leaf litter on the pond bottom and sedimented mud was noted. The microfauna in the litter on the pond bottom had a higher diversity than in the water or the sedimented mud. Patterns of seasonal change in the density of organisms — with one or two peaks in a year — were recognized in some taxa of protozoa and micrometazoa. Most peaks of bacterivorous protozoa in the leaf litter appeared in late autumn and spring. This phenomenon is considered to be closely related to the litter decomposition, because reduction of dissolved oxygen and/ or high bacterial density were observed in these two seasons. Nineteen species of protozoa from this pond were cultured in vitro with fallen leaves. From the results of the cultures and the observation of food vacuoles of protozoa in situ, part of the food web of the microfauna in the pond was estimated.     

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.     

Evaluation of subsampling and fixation procedures used for counting rumen protozoa.

Total numbers of protozoa can be significantly lower in rumen fluid than in whole rumen contents, depending on the time of sampling and the procedure used to separate the fluid and solid fractions. Moreover, generic distribution in rumen fluid was significantly affected in all cases tested. The percentage of Entodinium spp. increased, whereas percentages of Diplodinium spp. and Ophryoscolex spp. decreased. Microscopic observation of fresh and fixed rumen contents did not indicate any marked attachment of protozoa to particulate matter. In addition, dilution of whole rumen contents with water, 5 mM sucrose, or 0.1% Tween 80 before fixation did not affect total numbers or generic composition of protozoa. It was thus concluded that attachment to feed particles is probably not a problem in counting procedures. Blending of whole rumen contents to facilitate subsampling caused a decrease in numbers of protozoa. The concentration of formaldehyde used for preservation of rumen contents, 4, 10, or 18.5%, did not affect the total count.     

Abundance and productivity of protozoa in chalk streams

The distribution and abundance of protozoa in two chalk streams were studied. Particular attention was given to the ciliates and amoebae associated with the dominant submerged macrophyte, Ranunculus penicillatus . Highest numbers of protozoa occurred in habitats in the stream where the current velocity was reduced. Mean standing crops of ciliates, flagellates and amoebae were 32.5, 2.9 and 2.0 mg dry wt m⁻² of stream bed respectively. Estimates of production and respiration rates by field populations of ciliates and amoebae were made for one type of habitat within the streams. The results are compared with published data on the productivity of protozoa in other habitats and of other invertebrates within the chalk stream ecosystem. The limitations of such productivity estimates are discussed.     

Stabilité biologique des réseaux de distribution d'eau potable

The maintenance of the quality of water from the outlet of the treatment plant to the consumer tap is a major concern of water distributors. From a biological point of view, this maintenance must be characterized by a stability of biological features, namely bacterial growth from biodegradable organic matter, and protozoan bacterivory which must be not detectable. However, drinking water distribution systems are continuously exposed to a flow of biodegradable organic matter, which can represent around 20–30 % of the total dissolved organic carbon, and a flow of allochthonous microorganisms (bacteria, fungi, protozoa…), coming from the water treatment plant but also from incidents (breaks/repairs) on the distribution network itself. Apart from these microorganisms (heterotrophic bacteria in particular) can grow in this ultra-oligotrophic environment and colonize the all drinking water distribution system. The highest density of microorganisms occurs on the surface of pipewalls where they are organized in microcolonies (biofilm) that are mixed with corrosion products and inorganic precipitates. Five groups of organisms have been identified in distribution networks, in both the water phase and the biofilm: bacterial cells, protozoa, yeast, fungi and algae. The majority of these organisms are not pathogens, nevertheless potentially pathogen bacteria (Legionella…), fecal bacteria (coliforms, E. coli…), and pathogen protozoan cysts (Giardia intestinalis, Cryptosporidium parvum…) can transitorily find favorable conditions for their proliferation in the networks. Bacteria grow from the biodegradable fraction of dissolved organic matter while protozoa grow from dissolved organic matter, other protozoa but especially from bacterial prey items. The protozoan bacterivory was extensively studied in marine aquatic environments and in rivers, lakes,… but very rarely in drinking water distribution networks. Actually, proofs of the protozoan grazing on fixed and free-living bacterial cells were given by photography or film of biofilms accumulation on coupons that were previously immersed in potable water or by direct microscopic observation of bacteria in food vacuole of protozoa from potable water. A single and recent study has estimated protozoan bacterivory rate from laboratory experiences using fluorescent markers. It appears that in an experimental distribution system fed with biologically treated water (ozone/filtration through granular activated carbon), only ciliates present in the biofilm have a measurable grazing activity, estimated at 2 bacteria·ciliate⁻¹·h⁻¹ on average.Bacterial dynamics in drinking water distribution systems is complex and related to different parameters, like the biodegradable fraction of dissolved organic carbon, the presence of a residual of disinfectant, the nature and the state of pipewalls, the relative biomass of free and fixed bacterial, and grazing impact.The preservation of the biological stability of potable water during its storage in reservoir or its transport through the distribution systems can be achieved by (a) the use of chemical disinfectants (in particular by addition of chlorine) which is the widely used technique, or (b) the use of new techniques such as nanofiltration that can eliminate bacteria and significantly decrease the concentrations of organic matter at the inlet of the distribution network and in the potable water.

• (a)The use of oxidant, usually chlorine, induces a number of problems, in particular the development of oxidation by-products like trihalomethans (THM), among which some are recognized as carcinogenic products for animals. In addition, chlorine added at the outlet of treatment plant is consumed in the network and the maintenance of a residual of chlorine along an entire distribution network would need high concentrations of chlorine at the outlet of the treatment plant. This may be incompatible with standards for both residual chlorine and its by-products. Nevertheless, chlorine has a disinfectant effect on planctonic bacteria, if considering that only around 10 % of free bacterial cells are living cells, i.e. are able of respiratory oxidation. However, some studies show that bacteria fixed on granular activated carbon particles can be resistant to chlorine, as well as bacteria in aggregates. Thus, the addition of chlorine in potable water does not inhibit the formation of a biofilm at the surface of pipewalls. In the same way, protozoa transported by potable water can resist to chlorine.
• (b)The above disadvantages permitted the development of membrane filtration techniques like the nanofiltration, which is at the junction between reverse osmosis and ultrafiltration, and which seems to be an interesting alternative to conventional treatments because it presents the advantage to (i) decrease very strongly the concentrations of dissolved organic carbon (on average 90 % for DOC (Dissolved Organic Carbon) and 99 % for BDOC (Biodegradable Dissolved Organic Carbon)), (ii) to remove a very high proportion of almost the entire microorganisms (99 %), precursors of chlorination by-products, and micropollutans, (iii) to decrease the musty flavor of water (2-fold) and (iv) to produce a water that needs low concentration of chlorine.

     

The efficacy of biocides and other chemical additives in cooling water systems in the control of amoebae

Aims:  In vitro experiments were undertaken to evaluate biocide formulations commonly used in cooling water systems against protozoa previously isolated from cooling towers. The investigations evaluated the efficacy of these formulations against amoebic cysts and trophozoites.
Methods and Results:  Laboratory challenges against protozoa isolated from cooling towers using chlorine, bromine and isothiazolinone biocides showed that all were effective after 4 h. The presence of molybdate and organic phosphates resulted in longer kill times for bromine and isothiazolinones. All treatments resulted in no detectable viable protozoa after 4 h of exposure.
Conclusions:  The chemical disinfection of planktonic protozoa in cooling water systems is strongly influenced by the residence time of the formulation and less so by its active constituent. Bromine and isothiazolinone formulations may require higher dosage of concentrations than currently practiced if used in conjunction with molybdate- and phosphate-based scale/corrosion inhibitors.
Significance and Impact of the Study:  Cooling water systems are complex microbial ecosystems in which predator–prey relationships play a key role in the dissemination of Legionella . This study demonstrated that at recommended dosing concentrations, biocides had species-specific effects on environmental isolates of amoebae that may act as reservoirs for Legionella multiplication in cooling water systems.     

对虾养殖池塘底质修复技术对原生动物群落结构的影响

在对虾养殖池塘水体中共检测到原生动物86种,其中纤毛虫48种;鞭毛虫25种,肉足虫13种.原生动物优势种主要由一些运动能力相对较弱或固着生长的种类组成,如褐砂壳虫(Difflugia avellana)、瓜形膜袋虫(Cyclidium citrullus)、透明鞘居虫(Vaginicola crystalline)、多态喇叭虫(Stentor polymorphrus)和钟虫(Vorticella sp.)等,这些原生动物有以腐质和藻类为食、耐污性较强的特点.结果表明:在养殖初期,使用底质修复技术的试验池塘原生动物群落结构恢复比对照塘快,PFU群集的原生动物种类数和密度比对照塘多,水质比对照塘好,嗜污性相对较强的一些纤毛虫如尾草履虫(Paramecium caudaium)、拟瘦尾虫(Paruroleptus spp.)等比对照塘出现晚.在随后的养殖过程中,使用底质修复技术的试验塘原生动物群落结构比对照塘稳定,原生动物种类数和密度起伏小,pH值、透明度变化幅度也小于对照池塘,没有发生病害,水质较对照塘好.     

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.