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Growth of Acanthamoeba castellanii and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey

Zoë L. Pickup, Roger Pickup, Jacqueline D. Parry
DOI: http://dx.doi.org/10.1111/j.1574-6941.2007.00346.x 264-272 First published online: 1 August 2007


The growth responses of two species of amoeba were evaluated in the presence of live, heat-killed and heat-killed/5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)-stained cells of Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Klebsiella ozaenae and Staphylococcus aureus. The specific growth rates of both species were significantly higher with live bacterial prey, the only exception being Hartmannella vermiformis feeding on S. aureus, for which growth rates were equivalent on all prey states. There was no significant difference between growth rates, yield or ingestion rates of amoebae feeding on heat-killed or heat-killed/stained bacterial cells, suggesting that it was the heat-killing process that influenced the amoeba−bacteria interaction. Pretreatment of prey cells had a greater influence on amoebic processing of Gram-negative bacteria compared with the Gram-positive bacterium, which appeared to be as a result of the former cells being more difficult to digest and/or losing their ability to deter amoebic ingestion. These antipredatory mechanisms included microcolony formation in P. aeruginosa, toxin production in K. ozaenae, and the presence of an intact capsule in K. aerogenes. E. coli and S. aureus did not appear to possess an antipredator mechanism, although intact cells of the S. aureus were observed in faecal pellets, suggesting that any antipredatory mechanism was occurring at the digestion stage.

  • protozoa
  • growth-rate
  • yield
  • ingestion-rate
  • DTAF
  • heat-killed


Surface-associated microbial communities, i.e. biofilms, form on any surface that has contact with liquid (Costerton et al., 1987), but there has been a long-held assumption that these attached cells are afforded refuge from predation, particularly by single-celled protozoa, as a result of the fact that the cells are embedded in an exopolymer matrix (Costerton et al., 1981). This assumption may hold true for the smaller flagellates (Matz et al., 2004a, b, 2005; Weitere et al., 2005), but it does not hold true for the larger ciliates and amoebae, which effectively ingest attached bacteria (Parry, 2004) even if the bacterial cells are embedded within an exopolymer matrix (Heaton et al., 2001; Weitere et al., 2005). In fact, amoebae are particularly well designed for exploiting attached prey because their feeding form, the trophozoite, can only move and feed whilst on a surface (Rogerson & Laybourn-Parry, 1992; Rogerson et al., 2003; Pickup et al., 2007a). Despite our knowledge of this, quantitative data on the obligate amoebic predation of attached prey are scarce, and the reports that have been published have used a range of techniques, making comparison between datasets difficult.

The form of the bacterial prey employed constitutes one of the main methodological differences between published studies on amoebic growth. Studies have used either live bacterial cells (Butler & Rogerson, 1996; Heaton et al., 2001; Bracha et al., 2002; Hauer & Rogerson, 2005; Huws et al., 2005; Weitere et al., 2005; Cowie & Hannah, 2006) or dead, and often stained, cells (Rogerson et al., 1996; Butler & Rogerson, 1997; Mayes et al., 1997; Zubkov & Sleigh, 1999; Stapleton et al., 2005). The use of fluorescently labelled bacteria (FLB; Sherr et al., 1987) is not limited to amoebic studies; indeed, they are commonly used at trace levels for determining the ingestion rates of planktonic flagellates and ciliates (Epstein & Shiaris, 1992; Cleven, 2004). However, the heat-killing and staining processes have always caused concern, as they have the potential to alter the bacterial cells to such an extent that the nature of the protozoan−prey interaction might be affected. Many workers have found that the use of FLB does not affect protozoan growth (Sherr et al., 1987; Carlough & Meyer, 1990; Monger & Landry, 1992; Rogerson et al., 1996; Butler & Rogerson, 1997), but others have demonstrated that protozoa discriminate between live and heat-killed/stained bacteria, with the former leading to increased ingestion rates and/or growth rates (Drozanski, 1963; John et al., 1989; Landry et al., 1991; Monger & Landry, 1992; González et al., 1993; Parry et al., 2001).

The heat-killing of bacterial cells results in a loss of motility, which is a feature known to affect protozoan ingestion rates if the bacterium is highly motile (Monger & Landry, 1992; Matz & Jürgens, 2005). However, the lack of this feature in heat-killed cells should be of little importance when studying amoebic grazing on attached, immobilized prey. More importantly, heat-killed cells might possess altered surface characteristics, which would disrupt direct cell−cell recognition processes (Matz et al., 2002b; Wilks & Sleigh, 2004), or they may be unable to develop grazing-resistant cellular forms, i.e. microcolonies, in response to the grazing pressure (Matz et al., 2002a, 2004a; Weitere et al., 2005). The heat-killing process may also result in the coagulation of the bacterial cytoplasm, which has been shown to render the cells more difficult to digest (Mehlis et al., 1990), or may prevent the release of heat-labile bacterial toxins upon digestion (Groscop & Brent, 1964; Qureshi et al., 1993; Wang & Ahearn, 1997; Greub & Raoult, 2004). In addition, heat-killed cells lack the ability to express any form of cell-to-cell signalling, which might be crucial in influencing protozoan−prey interactions, as studies have shown that bacteria have the ability to signal both positively and negatively to eukaryotic cells (Thomas & Allsopp, 1983; Joint et al., 2000). Indeed, amoebae have shown both positive and negative chemotactic responses to bacterial prey (Singh, 1945; Seravin & Orlovskaja, 1977; Schuster et al., 1993; Bottone et al., 1994; Firtel & Chung, 2000; Weitere et al., 2005).

This study examined the effect of bacterial prey state (live, heat-killed and heat-killed/stained) on the growth parameters of two amoebic species and tested the hypothesis that amoebae can discriminate between live and heat-killed/stained cells. The study also sought to determine whether the process of staining the bacterial cells affected amoebic growth parameters more than the heat-killing of the cells before staining.

Materials and methods


The amoebae Acanthamoeba castellanii [Culture Collection of Algae and Protozoa (CCAP) 1501/1A] and Hartmannella vermiformis (CCAP 1524/7A) were routinely maintained on amoeba saline (AS) broth (Page, 1988), solidified with 1.5% agar (Lab M, Agar No2), and supplemented with a streak of Escherichia coli K12 10214 (source CCAP) at 20°C. Ten 6-day-old agar plates were examined microscopically for the presence of cysts, and these areas of agar, together with any remaining streak of E. coli, were removed prior to flooding the plates with AS broth, dislodging the attached amoebic cells with a sterile spreader, and collecting the suspension in a 40-mL tissue-culture flask. The amoebae were rested overnight at 20°C, and then the overlying AS broth was carefully removed and replaced three times with fresh AS broth. The flasks were gently vortexed for 20 s to dislodge the cells into suspension. A subsample of amoeba suspension was fixed with glutaraldehyde (1% v/v final concentration), and the concentration of amoebic cells was determined using a haemocytometer while that of E. coli cells (background bacteria) was determined using epifluorescence microscopy (see below). This resulted in amoebic suspensions containing c. 104E. coli cells mL−1.

Bacterial prey

Five bacterial species that have been previously shown to support a positive growth rate of the two species of amoeba (Pickup et al., 2007b) were employed in this study. Escherichia coli K12 10214 (source CCAP, UK), Pseudomonas aeruginosa SG 81 (source H.-C. Fleming, Gerhard Mercator University, Germany), Klebsiella aerogenes [National Collection of Type Cultures (NCTC) 9528], Klebsiella ozaenae (isolated by J. English, Lancaster University, UK) and Staphylococcus aureus (NCTC 6571) were routinely maintained as streaks on nutrient agar plates incubated at 25°C. Cell suspensions were prepared in sterile distilled water after 24 h of growth. One-third of each suspension was subjected to a temperature of 60°C for 2 h in a water bath (‘heat-killed cells’), another third was heat-killed, as above, in the presence of 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF) and subsequently washed following Sherr et al. (1987) to generate ‘stained cells’, and the remaining third was left untouched (‘live cells’). All suspensions were stored at 4°C for no longer than 2 days and then sonicated for 10 min before use, using a Camlab Transonic T460 ultrasonic water bath (Camlab, Cambridge, UK). Cell concentration was determined on a subsample fixed with glutaraldehyde (0.4% v/v final concentration). The live and heat-killed cells were stained with 4′,6-Diamidino-2-phenylindole (DAPI) (Porter & Feig, 1980) before filtration onto 0.2-μm pore-size black track-etched polycarbonate filters (Osmonics, GE Water & Process technologies, Philadelphia) and viewing with a Leitz Labourlux S epifluorescent microscope at a final magnification of × 1250 under UV excitation (BP340/LP430 filter block). The DTAF-stained cells were not DAPI stained, but were observed as yellow fluorescent cells under blue excitation (BP450/LP490 filter block). At least 400 cells per filter were counted before conversion to cells mL−1.

Grazing experiments

AS broth was solidified with 1.5% agarose (Sigma), and 90-mm Petri dishes were filled with 45 mL of molten agar to yield an agar depth of 7 mm. Agarose has previously been shown to limit the growth of live bacterial cells on the agar surface during grazing experiments (Pickup et al., 2007b). Each species of amoeba was challenged with five species of bacterial prey in each of the three states (live, heat-killed and stained). For each combination, the amoeba species and bacterial prey were coinoculated into 2 mL of AS broth, which, when poured onto the agar surface and allowed to dry in a laminar flow cabinet, resulted in the desired starting concentrations as cells cm−2 agar. The starting concentrations for bacteria and amoebae were 5 × 107 and 5 cells cm−2, respectively. Once the agar surfaces had dried, the resultant lawns were examined microscopically for 1-cm2 areas showing confluent bacterial lawns and the presence of about 5 amoeba cells. Three 1-cm2 areas of inoculated agar were aseptically excised from the Petri dish, and each was placed onto 10 μL of sterile distilled water in a Sedgwick Rafter Counting Chamber. Controls of each bacterial species alone, and each species of amoeba alone, were also prepared in triplicate. The counting chambers were incubated at 20°C, on top of moistened foam in perspex boxes, to prevent desiccation. The agar sections were examined twice a day with a light microscope (× 400 to × 1200 final magnification, depending on species) for up to 5 days, and the number of amoebae on the 1-cm2 area was recorded. Any unusual amoebic behaviour, or cyst formation, was also recorded. At the beginning and end of the experiment, additional agar surfaces of tests and bacterial controls were fixed in glutaraldehyde (0.4% v/v final concentration), and the surfaces were scraped to remove the adherent cells into suspension. The bacterial concentration (cells cm−2) was determined by epifluorescence microscopy, as previously described.


All analyses were carried out using spss statistics (SPSS Inc., IL), sigmaplot graphics (SPSS Inc.) and Microsoft Excel (Microsoft, Redmond, WA). The specific growth rate (μ, h−1) of the amoebae on each prey treatment was determined by regression analysis of the linear portion of the graph of ln(amoeba cells cm−2) against time (h), generally over a 3-day period, after correcting for any amoebic growth in the control systems. A one-way between-groups ANOVA was conducted to explore the effect of heat-killing and staining of prey on specific growth rate. Post hoc tests were conducted to examine the significance of any between-groups effect. Ingestion rate (prey amoeba−1h−1) was determined by interpolating bacterial concentration at the midpoint of the whole experimental period, after correcting for the bacterial control, and dividing this by the average concentration of amoebae following Sherr et al. (1983). The number of amoeba cells produced per ingested prey cell (yield) was determined by dividing the number of new amoebae produced in the system by the number of bacteria removed from the system. Ingestion rates and yields were compared using a Student's t-test.


Figure 1 shows the calculated values for specific growth rates, and Table 1 the calculated ingestion rates and yields, for H. vermiformis and A. castellanii feeding on five bacterial species in three cellular states (live, heat-killed and stained). The specific growth rates of H. vermiformis were significantly higher on the live cells of E. coli (P<0.02) and P. aeruginosa, K. aerogenes and K. ozaenae (P<0.01) compared with those on the heat-killed or stained cells, which were not significantly different from each other (Fig. 1). However, the form of S. aureus did not influence the calculated specific growth rates of this species of amoeba (Fig. 1g). The specific growth rates of A. castellanii were significantly higher on the live cells of E. coli, P. aeruginosa, K. aerogenes (P<0.01) and S. aureus (P<0.05) compared with those on the heat-killed and stained cells, which were not significantly different from each other (Fig. 1). This amoeba had a significantly lower specific growth rate on heat-killed K. ozaenae (P<0.01) compared with the rates on the live and stained cells, which were equivalent (Fig. 1j).


Calculated specific growth rates (μ, h−1) (±SEM) for Hartmannella vermiformis and Acanthamoeba castellanii feeding on 5 × 107 cells cm−2 of live (L), heat-killed (HK) and DTAF-stained (S) cells of Escherichia coli (a and b), Pseudomonas aeruginosa (c and d), Klebsiella aerogenes (e and f), Staphylococcus aureus (g and h), and Klebsiella ozaenae (i and j), at 20°C.

View this table:

Calculated ingestion rates (IR, prey cells amoeba−1h−1) and yields (Y, × 10−3 amoeba per prey cell) (± SEM) for Hartmannella vermiformis and Acanthamoeba castellanii feeding on live, heat-killed and DTAF-stained Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Staphylococcus aureus and Klebsiella ozaenae on an agar surface at 20°C

PreyH. vermiformisA. castellanii
E. coli K12
Live265 (80)0.0953 (0.033)265 (126)0.195 (0.08)
Heat-killed119 (45)0.1350 (0.059)656 (98)*0.035 (0.003)
Stained219 (103)0.0945 (0.033)203 (53)0.127 (0.033)
P. aeruginosa
Live283 (65)0.0818 (0.020)318 (104)0.110 (0.031)
Heat-killed418 (352)*0.0169 (0.007)*939 (289)*0.011 (0.006)*
Stained980 (713)*0.0035 (0.003)*861 (24)*0.012 (0.004)*
K. aerogenes
Live250 (20)0.1023 (0.008)188 (129)0.534 (0.343)
Heat-killed693 (100)*0.0158 (0.003)311 (133)*0.125 (0.077)
Stained445 (188)*0.0638 (0.037)365 (65)*0.032 (0.004)
S. aureus
Live574 (199)0.0408 (0.019)686 (144)0.042 (0.007)
Heat-killedNDND234 (88)*0.045 (0.008)
Stained355 (196)0.0677 (0.025)172 (62)*0.100 (0.041)*
K. ozaenae
Live395 (227)0.0818 (0.027)195 (138)ND**
Heat-killed365 (19)0.0349 (0.003)ND*ND*
Stained344 (94)0.005 (0.001)*259 (113)ND**
  • ND, samples lost; ND*, negative growth rates prevented the calculation of IR and Y; ND**, minimal growth rates provided meaningless Y values, which were thus omitted from the analysis.

  • Significantly different from the value obtained with

  • * Live cells,

  • Stained cells, or

  • Heat-killed cells.

The calculated yield values for both species of amoebae feeding on all forms of bacterial prey were, in general, equivalent. The only exceptions were with heat-killed P. aeruginosa cells, which gave lower yields for both species (P<0.01), stained K. ozaenae, which gave a lower yield for H. vermiformis (P<0.01), and stained S. aureus, which gave a higher yield for A. castellanii (P<0.05) (Table 1). The calculated ingestion rates showed the most variation. Both species of amoebae showed significantly lower uptake of the live cells of P. aeruginosa (P<0.01) and K. aerogenes (P<0.02), and equivalent rates with K. ozaenae (Table 1). Hartmannella vermiformis also ingested the live, heat-killed and stained cells of E. coli and S. aureus at equivalent rates (Table 1), but A. castellanii ingested more heat-killed E. coli cells than live (P<0.01), and more live S. aureus cells than heat-killed or stained (P<0.05) (Table 1).

The trophozoites of both species of amoebae began feeding and dividing immediately on the lawns of all bacteria except in the presence of K. ozaenae. This bacterium induced unusual amoebic behaviour, even after the prey cells had been heat-killed (with or without staining). Unlike the normal trophozoite response, which was to move over the lawn in a semi-random manner (the Levy walk, Levandowsky et al., 1997), the presence of live, heat-killed or stained K. ozaenae caused an immediate cessation in amoebic movement. The trophozoites of H. vermiformis remained stationary for a period of 26 h, after which normal feeding and movement was resumed. In the presence of live K. ozaenae, the trophozoites of A. castellanii remained stationary for 14 h, after which they rapidly moved in a straight line off the agar surface, or attempted to bury themselves in the agar as observed in a previous study (Pickup et al., 2007b). In the presence of heat-killed and stained K. ozaenae the trophozoites remained stationary for the duration of the experiment. No cysts were produced by the A. castellanii populations in the presence of K. ozaenae; instead, many of the trophozoites lysed.


The growth responses of two species of amoebae were evaluated in the presence of live, heat-killed and (heat-killed) stained cells of five bacterial species that are known to exist within natural biofilms (Costerton et al., 1987; McBain et al., 2003). Results showed that, in general, both species achieved significantly higher specific growth rates when feeding on live bacterial cells and there was no significant difference between amoebic parameter values obtained with heat-killed or (heat-killed) stained bacterial cells. This indicates that it was the heat-killing process, and not the subsequent staining, that affected the amoeba−bacterial interaction.

Higher growth rates and yields of amoebae with live bacterial cells, compared with their heat-killed counterparts, have been recorded previously (Drozanski, 1963; John et al., 1989). However, other studies have shown that heat-killing and staining has no effect on growth rate, or that rates are higher with the stained prey cells (Rogerson et al., 1996; Butler & Rogerson, 1997). The variability in published data might be related to the Gram reaction of the bacterium employed. In the present study, all amoebic specific growth rates on live cells of the Gram-negative bacteria (E. coli, P. aeruginosa, K. aerogenes and K. ozaenae) were higher than those achieved with their heat-killed or stained counterparts, agreeing with Drozanski (1963) and John et al. (1989), who also employed Gram-negative bacteria (Aeromonas aerogenes and E. coli). In contrast, the amoebic specific growth rates achieved with live cells of the Gram-positive bacterium S. aureus were more variable. For example, no significant difference in specific growth rates was recorded for H. vermiformis feeding on the three states of this prey, which agrees with Rogerson et al. (1996) and Butler & Rogerson (1997), who also employed a Gram-positive bacterium (Planococcus citreus). Thus, it appears that the heat-killing process affects amoebic interactions with Gram-negative bacteria more than it does those with Gram-positive bacteria, but examination of specific growth rates alone cannot discern whether the heat-killing process has predominantly affected the ingestion or subsequent digestion of these prey.

There are currently no published data on the susceptibility of different bacterial species to amoebic digestion, but some insight can be obtained from amoebic yield. The majority of data show that higher amoebic yields are obtained upon the digestion of live Gram-negative prey, suggesting that heat-killed Gram-negative cells are more difficult to digest. Amoebic yields in this study were not elevated with the live cells of the Gram-positive S. aureus, suggesting that heat-killing did not affect the digestibility of these cells. This contradicts the results of Mehlis et al. (1990), who showed that the coagulated cytoplasm of heat-killed S. aureus was more difficult to digest than live cytoplasm by the ciliate Paramecium; a plausible reason for this discrepancy cannot be provided at present. However, the similarity in the responses of the two species of amoebae, with each of the prey, does suggest that it is the properties of the bacterial prey, and not of the amoebae, which govern the nature of the amoeba−bacterial interaction, and this was further highlighted upon examination of the ingestion-rate data.

Amoebic ingestion rates on live vs. heat-killed or stained bacterial cells split the amoeba−bacterium combinations into two distinct groupings, namely those combinations for which the ingestion of live bacterial cells was lower than for their heat-killed or stained counterparts (P. aeruginosa and K. aerogenes) and those for which it was not (E. coli, S. aureus and K. ozaenae). Live cells of P. aeruginosa and K. aerogenes are known to be acceptable prey for these two species of amoebae (Weekers et al., 1993; Pickup et al., 2007b), but heat-killing these cells produced a significant reduction in amoebic growth rates (Fig. 1), particularly when the prey was P. aeruginosa (Fig. 1c and d). Heat-killed or stained bacteria were ingested at higher rates, by both species of amoeba, compared with their live counterparts, but the amoebic yields were lower (significant for P. aeruginosa only) (Table 1), re-iterating the fact that heat-killing makes these cells more difficult to digest. Digestion of the live counterparts appeared more effective, with the yield from K. aerogenes being the highest, and that from P. aeruginosa the third highest, out of all five bacterial species (Table 1). Data on the digestibility of these bacterial genera in protozoa are rare. Only one study has shown that P. fluorescens is digested at a faster rate than A. hydrophila in the flagellates Bodo saltans and Goniomonas sp., but not in the ciliate Cyclidium glaucoma (Jezbera et al., 2005). The higher yields, and specific growth rates, recorded with live cells of P. aeruginosa and K. aerogenes were achieved upon the ingestion of fewer cells, compared with their heat-killed/stained counterparts. This suggests that live cells may also possess a mechanism with which to deter predation, which is lost upon heat-killing.

Live P. aeruginosa cells were observed to develop microcolonies on the surface of the agar plate during the course of the experiment, as has been described previously (Matz et al., 2004a; Weitere et al., 2005). The amoebae were clearly visible exploiting these areas of prey, but the aggregated form of this prey may have been the cause for the observed reduction in ingestion rates. Pseudomonas species are not limited to the formation of microcolonies as a defence mechanism, as some strains can be toxic to amoebae (Singh, 1945; Groscop & Brent, 1964; Qureshi et al., 1993; Weitere et al., 2005). Live cells of K. aerogenes are not known to be toxic to protozoa, and the cells did not exhibit microcolony formation within the experimental period. It is therefore postulated that the reduced affinity shown for live K. aerogenes cells by the amoebae is a result of the presence of the extracellular polysaccharide capsule of Klebsiella species in general. This capsule has been implicated in providing resistance to phagocytosis and/or digestion by mammalian white blood cells (Allen et al., 1987), but no data regarding the effectiveness of the capsule in deterring amoebic grazing are available.

Several species of Hartmannella and Acanthamoeba have been observed ingesting and surviving on S. aureus (Groscop & Brent, 1964; Huws et al., 2005), but Gram-positive bacteria are considered inferior prey for protozoa (Taylor & Berger, 1976; González et al., 1990). In the present study, this bacterium provided both species of amoebae with their lowest yield, even though the prey cells were ingested at the highest rate (Table 1). This suggests that S. aureus possesses no mechanism for deterring ingestion by amoebae, possibly because it possesses post-ingestion defences such as a thicker cell wall, or an anti-oxidant yellow carotenoid, which has been shown to protect the cells from destruction by white blood cells (Liu et al., 2005). Whatever the mechanism, faecal pellets containing intact S. aureus cells were observed during the experiments, indicating that only a proportion of the ingested S. aureus cells were digested within one passage through the cells of both amoebae species. However, partial digestion may only be a response of this particular strain, as other, more pathogenic, strains have been shown to invade amoebic cells and to exist within the cytoplasm (Huws et al., 2006).

Escherichia coli K12 gave the second highest yield values for both species of amoeba (Table 1), which demonstrates its highly digestible nature (González et al., 1990). Indeed, Arana et al. (2003) found that live E. coli cells were far less resistant to protozoan grazing compared with the other endogenous bacteria within river biofilms. The heat-killing of the cells resulted in significantly lower specific growth rates in both species of amoeba (Fig. 1a and b), but the mechanism(s) by which this was achieved could not be discerned from the dataset. The data did suggest that E. coli K12 possesses no mechanisms for deterring ingestion or reducing digestion, but this result may have been down to the strain used. For example, E. coli K12 has been observed to be digested by the ciliate Tetrahymena pyriformis (J. Parry, unpublished data), but 90% of ingested E. coli HB101 cells have been shown to survive a single passage through this ciliate (Schlimme et al., 1997). In addition, the highly pathogenic E. coli O157 has been shown to survive and replicate within cells of Acanthamoeba polyphaga (Barker et al., 1999).

Klebsiella ozaenae, a capsulated, heavily pigmented red subspecies of K. pneumoniae, gave the second lowest yield in both species of amoeba (Table 1). The heat-killing of the cells resulted in no significant effect on ingestion rate, suggesting that the bacterium possessed no mechanism for deterring ingestion. However, the presence of a lag phase in the amoebic growth response, and visual observation of the behaviour of the trophozoites, showed this not to be true. The presence of live cells arrested the movement of H. vermiformis and A. castellanii for 26 and 14 h, respectively, after which normal movement and grazing were resumed in the H. vermiformis population only. Indeed, this amoeba has previously been shown to respond to another pigmented bacterium, Serratia marcescens, in a similar manner (Groscop & Brent, 1964). Despite this slow start, the specific growth rate of H. vermiformis was highest on these live cells, followed by on the heat-killed cells (P<0.05), and then on the stained cells (P<0.05). This trend was also evident with the yield values, although a significant difference was only recorded with stained cells (Table 1). These results, coupled with no significant difference in ingestion rate, suggest that the heat-killing of these cells either made the cells more difficult to digest or altered them in some way so as to release an unknown factor from the cells after death. Acanthamoeba castellanii was particularly sensitive to K. ozaenae in all three states. The trophozoites did not resume normal motility in the presence of heat-killed or stained cells but remained stationary, with a number of cells lysing. Thus, the presence of heat-killed cells prevented the induction of cyst formation in this amoeba, and suggests that the heat-killing was affecting the preingestion stage of the amoeba−bacterial interaction and not the digestion stage. In the presence of live cells, the trophozoites did not resume normal movement and grazing, and either attempted to leave the agar surface or lysed; thus, the low specific growth rates may be a result of both cell death and emigration.

In conclusion, the results of this study suggest that the Gram-positive S. aureus does not appear to invest energy into an anti-predatory defence mechanism, possibly because a proportion of the ingested cells can survive a passage through the amoebic cells. However, most of the Gram-negative bacteria possess a mechanism with which to deter amoebic ingestion, but excessive heat destroys this and appears to render the bacterial cells easier to ingest but more difficult to digest. These few examples highlight the complexity of amoebae−bacteria interactions, with the bacterial prey driving the nature of the interaction, possibly by means of cell-to-cell signalling (Davies et al., 1998). Of all protozoa, amoebae should show a heightened response to signal molecules because of their obligate surface-associated life-style, having no escape from signals whether positive or negative. Conversely, the more transient flagellates and ciliates could visit the biofilm intermittently, allowing them some refuge from any toxic signal (Weitere et al., 2005). Thus more information regarding the factors influencing the nature of amoebae−bacteria interactions, the digestibility of different prey, and the growth response of the amoebae is called for in order to unravel the nature of these microbial predatory interactions in natural biofilm habitats.


The authors would like to thank Dave Montagnes for his advice regarding the statistical analysis of the data.


  • Editor: Riks Laanbroek


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