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Keywords:

  • pathogen;
  • Staphylococcus aureus;
  • Acanthamoeba polyphaga;
  • amoebae;
  • protozoa–bacteria interactions

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Grazing by heterotrophic protists is a major source of bacterial mortality in soil (Clarholm, 1981; Ekelund et al., 2002), marine (Barbaree et al., 1986; Epstein & Shiaris, 1992) and freshwater (Sanders et al., 1989; Iriberri et al., 1995) systems and is considered to be a major trophic pathway whereby the biomass produced by bacteria, cyanobacteria and algae re-enters the food web (Azam et al., 1983).

Nonetheless, not all bacteria are ingested and digested by protozoa, some escape protozoan ingestion for a number of reasons, including their cell size (Šimek & Chrzanowski, 1992; Jürgens & DeMott, 1995; Kinner et al., 1998; Posch et al., 1999). Some are ingested but have evolved strategies of evading digestion and even multiplying within protozoa, the prototypical example being Legionella pneumphila (Rowbotham, 1980; Barbaree et al., 1986; Brown & Barker, 1994; Brown & Barker, 1999; Molmeret et al., 2005). It is also frequently reported that L. pneumophila uses similar mechanisms to invade protozoan and mammalian cell lines (Segal & Shuman, 1999; Gao & Abu Kwaik, 2000; Molmeret et al., 2005); thus protozoa have been termed as ‘trojan horses’ (Brown & Barker, 1994) or ‘biological gymnasia’ whereby intraprotozoan cells train for their encounter with the more evolved mammalian cells (Harb et al., 2000). There is also increasing evidence suggesting that other bacterial species, for example Burkholderia cepacia (Marolda et al., 1999), Chlamydophila pneumoniae (Essig et al., 1997; Fritsche et al., 2000; Poppert et al., 2002), Escherichia coli 0157 (Barker et al., 1999), Mycobacterium avium (Cirillo et al., 1997; Steinert et al., 1998), and methicillin-resistant Staphylococcus aureus (MRSA; Huws et al., 2006) can also evade digestion within amoebae. Thus, amoebae may augment the survival of many pathogenic bacteria within the environment.

Although data are increasing with regard to certain bacteria being able to survive digestion within protozoa, data are still sparse and currently available data are sometimes conflicting. Here we have investigated whether the survival of Bacillus cereus, Enterococcus faecalis, Enteropathogenic E. coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive S. aureus (MSSA) is augmented by the presence of Acanthamoeba polyphaga. Acanthamoeba polyphaga is ubiquitous in soil (Rodriguez-Zaragoza, 1994), aquatic (Hunt & Parry, 1998), and healthcare (Rohr et al., 1998) environments and so it will commonly encounter these pathogens in nature.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Bacteria and their growth conditions

Bacillus cereus ATCC 14579, E. faecalis JH2-2, EPEC E2348/69, L. monocytogenes NCTC 11994 (serovar 4b), S. enterica serovar Typhimurium SL1344, and MSSA RN4220 were used throughout. All bacteria were grown to stationary phase (16 h) in Luria–Bertani (LB) broth at 37 °C, shaken at 100 r.p.m. Viable bacteria were counted on LB agar following serial dilution and incubation at 37 °C for 24 h. For coculture experiments, bacteria were harvested by centrifugation (10 000 g, 5 min) and washed twice in Neff's amoebal saline (NAS; Page, 1976), before OD adjustment to the required cell density.

Amoebae and their growth conditions

Acanthamoeba polyphaga was obtained from Dr T.J. Rowbotham (Public Health Laboratories, Leeds, UK) and grown in proteose peptone-yeast-glucose (PYG) medium as monolayers in 75 cm2 tissue culture flasks at 23 °C. Amoebae were subcultured weekly by gently tapping flasks to detach cells before diluting 1 : 10 in fresh medium. Stationary phase (3–5 days) cultures were used throughout this study. For coculture experiments amoebae were harvested by gently tapping flasks, followed by centrifugation (300 g, 5 min) and washing twice in NAS, before numbers were adjusted to c. 2 × 105 cells mL−1 using a haemocytometer.

Coculture experiments

Cocultures were prepared in 12-well plates (Helena Biosciences, Sunderland, UK). Wells were seeded with A. polyphaga (c. 2 × 105 cells mL−1) and amoebae were allowed to adhere for 30–60 min before the addition of bacteria at MOI (Multiplicity of Infection) 1 : 100 or 1 : 1000 of amoeba to bacteria. Plates were incubated at 37 °C, and following various time intervals, wells were cell-scraped, with dilution of the resulting cell suspension in saline (0.9% w/v) and colony counts of extracellular bacteria determined. Control bacterial and amoebal monocultures were also set up. Viability of A. polyphaga was assessed using the trypan blue exclusion assay (Gao et al., 1997).

Intracellular growth kinetics

Cocultures were prepared as described, with L. monocytogenes and S. aureus, however the microplates were centrifuged (300 g, 5 min, 37 °C) following the addition of bacteria to enhance contact between bacteria and adherent amoebae. After 2 h, the extracellular medium was aspirated and the attached amoebal layer was washed twice in NAS. Gentamicin sulphate (50 mg L−1 for 2 h), in the case of L. monocytogenes, or lysostaphin (20 mg L−1 for 2 h), in the case of S. aureus, was added to kill c. 99.99% of extracellular bacteria. The antimicrobials were removed by washing twice with NAS (designated time 0 h), after which intracellular growth was followed using the method of Gao et al. (1997). Briefly, growth kinetics were determined by adding the number of released bacteria to the number of intracellular bacteria released following lysis of amoebae for c. 5 min with cold triton X-100 (0.1% v/v).

Growth in coculture cell-free supernatant

Cocultures were prepared as described with L. monocytogenes and S. aureus in 12-well microplates. Following 12 h (S. aureus) or 24 h (L. monocytogenes) of coculture, wells were cell-scraped and the contents were filter sterilized through 0.2-μm pore size filters. Cell-free medium was then inoculated with stationary phase, NAS washed, bacterial cells at a density of 2 × 107 CFU mL−1 (as described for coculture experiments). Plates were incubated at 37 °C, and following various time intervals, wells were cell-scraped, with dilution of the resulting cell suspension in saline (0.9% w/v) and colony counts of bacteria determined.

Localization of intracellular bacteria

To visualize intracellular bacteria, cocultures were prepared as described, using bacteria that had been prestained with 10 μM Cell Tracker BODIFY Viable Green for 30–60 min according to the manufacturer's recommendations (Molecular Probes, Leiden, The Netherlands). We chose the highest concentration of dye that did not affect the extracellular numbers of the bacteria in the presence and absence of amoeba at the concentration used (data not shown). This was done as the stain concentration halves during each doubling as it passes to the daughter cells. Regardless, following 24 h of coculture, amoebae were harvested by cell-scraping followed by centrifugation and suspended in 5 nM Lyso Tracker Red for 30–60 min as recommended by the manufacturer (Molecular Probes). Lyso Tracker red stains acidic organelles red. Cells were dried on poly-l-lysine slides before visualization under a confocal microscope (Zeiss Axiovert 100 M, Carl Zeiss Vision, Oberkochen, Germany). The occurrence of intracellular bacteria was quantified by scanning 100 fields of view (overall magnification × 100) and expressing their occurrences as percentage in terms of total amoebae viewed.

Statistical analysis

Normality of extracellular bacterial counts in the presence and absence of A. polyphaga was ascertained before anova was performed on the data sets (Log10 transformed). A Bonferroni post hoc test was performed to determine significance between control and test data at each time point. Normality of amoebal survival data in the presence and absence of bacteria was also ascertained before data were transformed (Log10) and t-tests performed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

Cointeractions of bacteria and amoebae

In our coculture system we followed the extracellular bacterial viable count and compared it with the survival of bacteria grown without amoebae. If the comparative counts in the presence of amoebae were lower then this was evidence of predation. If counts were higher then amoebae were sustaining bacterial survival and we sought to determine whether there was intra-amoebal growth (and release), extracellular bacterial growth on amoebal products or a combination of both. Figure 1a–f, illustrates the effects of A. polyphaga on extracellular bacterial numbers. All bacteria, except L. monocytogenes (Fig. 1e) and MSSA (Fig. 1f), were significantly (P<0.05) lower in numbers in the presence of the amoebae compared with controls in the absence of amoebae. Extracellular numbers of B. cereus (Fig. 1a) and E. faecalis (Fig. 1b) decreased by over 1 log cycle after 72 h of coculture, as did EPEC (Fig. 1c) and S. enterica serovar Typhimurium (Fig. 1d). Survival of L. monocytogenes (Fig. 1e) and MSSA (Fig. 1f) decreased more than 10 000-fold after 72 h; however, in the presence of amoebae, numbers were at least 3 log cycles higher, indicating that some amoebal interaction was enhancing bacterial survival. There were no significant differences (P>0.05) in amoebal viability in the various bacterial cocultures compared with amoebae alone (Fig. 2). Nonetheless it was apparent that in the presence of EPEC, B. cereus and S. typhimurium, amoebae had grown by c. 160%, 150% and 120%, respectively, compared to in the absence of these bacteria, indicative of ingestion and digestion. In the presence of L. monocytogenes, E. faecalis and S. aureus, amoebae were c. 90%, 80% and 70%, respectively, lower in number, compared with in the absence of these bacteria, which suggests that they were not good food sources.

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Figure 1.  Extracellular numbers of (a), Bacillus cereus 14 579; (b), Enterococcus faecalis JH2-2; (c), Enteropathogenic Escherichia coli E2348/69; (d), Salmonella enterica serovar Typhimurium SL1344; (e), Listeria monocytogenes NCTC 11994 and (f), Staphylococcus aureus RN4220 in the absence (closed symbols) and presence (open symbols) of Acanthamoeba polyphaga. Data are SE of the mean for at least three replicate experiments; *P>0.05.

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image

Figure 2.  Viability of Acanthamoeba polyphaga following 24 h of coincubation with the test bacteria. Results illustrate percentage survival relative to control (absence of bacteria) amoebal counts. Data are SE of the mean for at least three replicate experiments.

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Internalization and intracellular growth of L. monocytogenes and S. aureus

At time 0 h, A. polyphaga had internalized c. 4 × 103 listeriae per 106 amoebae; however, from 4 h onwards intracellular and released bacteria were undetectable (Fig. 3). In contrast, at time 0 h, A. polyphaga had internalized c. 2 × 105 MSSA per 106 amoebae and combined intracellular and released bacterial counts increased by c. 1 log cycle after 24 h (Fig. 3).

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Figure 3.  Intracellular growth kinetics of Listeria monocytogenes NCTC 11994 (•), and Staphylococcus aureus RN4220 (○) within Acanthamoeba polyphaga. Data are SE of the mean for five replicate experiments.

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Growth in coculture cell-free medium

It was evident that L. monocytogenes may be sustained by metabolic waste products released during coculture (Fig. 4a). Following 48 h in the presence of 24 h cell-free coculture medium, L. monocytogenes were c. 1 log higher compared to amoebal and bacterial control cell-free supernatants. The same was not apparent with S. aureus and numbers present in coculture cell-free supernatant were comparable to those present in amoebal and bacterial control cell-free supernatants (Fig. 4b).

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Figure 4.  Bacterial counts of Listeria monocytogenes (a) and Staphylococcus aureus (b) in the presence of coculture cell-free medium (•), amoebal control cell-free medium (▪), or bacterial control cell-free medium (▴). Data are SE of the mean for three replicate experiments.

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Localization of intracellular bacteria

Confocal microscopy showed that c. 30% of amoebae contained S. aureus within acidic vacuoles (Fig. 5a–d), and c. 10% of the amoebae were heavily infected with cocci throughout (Fig. 5e–h). Confocal microscopy also revealed that c. 30% of amoebae contained intravacuole L. monocytogenes, with most being within acidified vacuoles (Fig. 6a–d).

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Figure 5.  Confocal microscopy images of Staphylococcus aureus RN4220 within vacuoles (a–d; depth 1.24 μm) and heavily infected Acanthamoeba polyphaga (e–h; depth 5.60 μm). Staphylococcus aureus cells are stained green (b and f), and acidic vacoules are red (c and g) with the corresponding overlay (d and h). The corresponding light micrographs are shown (a and e). Scale bar=5 μm.

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image

Figure 6.  Confocal microscopy images of Listeria monocytogenes NCTC 11994 within vacuoles (a–d; depth 5.0 μm). Listeria monocytogenes cells are stained green (b), and acidic vacuoles are red (c) with the corresponding overlay (d). The corresponding light micrograph is shown (a). Scale bar=5 μm.

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Discussion

Our experimental aims within this study were to investigate the spectrum of pathogen–amoebal interactions under the same experimental conditions. This is the first time that interactions of amoebae have been looked at with regard to E. faecalis and B. cereus. However, we recognize that the predation/survival/intracellular replication data for the other bacteria presented here differ in some instances from some work already published. For example, we found that EPEC does not survive predation by A. polyphaga, whereas E. coli O157 does (Barker et al., 1999). Also, we found no evidence that S. enterica serovar Typhimurium survived in the presence of A. polyphaga under our test conditions, whereas others have shown that S. enterica serovar Typhimurium proliferates within A. rhysodes (Tezcan-Merdol et al., 2004) and A. polyphaga (Gaze et al., 2003). The lack of proliferation in our system may be due to differences in the bacterial and amoebal strain and the low MOI used by Tezcan-Merdol et al. (2004). Gaze et al. (2003), on the other hand, used the same bacterial and amoebal strains, but with an agar-based approach and temperatures of either 25 or 35 °C. Unusually, intracellular replication occurred only in contractile vacuoles and was seen in fewer than 1% of amoebae. We did not see growth in contractile vacuoles, nor did we see increases in extracellular Salmonellae numbers in our system. Additionally, Ly & Müller (1990) showed that following 8 days of coculture at 36 °C, viable listeriae were released from Acanthamoeba sp., before they encysted, with any remaining intracellular listeriae being killed within the cysts. We found that our strain of L. monocytogenes could not survive or replicate within A. polyphaga in our test system; however, extracellular numbers did increase, likely sustained on excreted amoebal products or debris. Indeed, our data agree with a recent report by Zhou et al. (2007), who looked at survival of various species of Listeria as well as many serovars of specifically L. monocytogenes within A. castellanii. They found that the presence of amoebae caused numbers of L. monocytogenes (serovars 4b, 1/2a, and 1/2c) to be higher than in the absence of amoebae. They also concluded that they found no evidence for intracellular replication of L. monocytogenes and that it was possible that the bacteria were scavenging naturally dead amoebae or that metabolic waste products were sustaining the growth of their bacteria over the duration of the coculture.

We have previously shown that many isolates of epidemic MRSA survive and potentially proliferate within A. polyphaga (Huws et al., 2006). Indeed, extracellular and intracellular bacterial numbers obtained in this study with a MSSA in the presence of amoebae were very similar to previous results with epidemic MRSA isolates 15 and 16. Thus, it is apparent that irrespective of their methicillin susceptibility, S. aureus have the capacity to survive within A. polyphaga. Recently published data also suggest that S. aureus avoid digestion within the amoebae Hartmannella vermiformis and A. castellanii (Pickup et al., 2007a, b). They suggest that S. aureus possess no specific mechanism for evading digestion but have postingestion defences such as a thicker cell wall, or an antioxidant yellow carotenoid. In this study fluorescent confocal microscopy revealed that after 24 h of coculture, c. 30% of amoebae contained viable cocci within phago-lysosomes, and c. 10% of amoebae had viable bacteria throughout the cytoplasm. So, we believe that S. aureus may be able to withstand acidic conditions and potentially accomplish intracellular replication. The heavily infected amoebae also stain red throughout the cell, providing evidence that perhaps phago-lysosomal fusion does occur before the bacterium escapes the phago-lysosome, thus also releasing the red stain to the cytoplasm. However, recent data investigating Burkholderia cepacia complex within A. polyphaga showed their intracellular survival through an alternative mechanism of survival, specifically within an acidified vacuole that was distinct from the lysosomal compartment (Lamothe et al., 2004). We cannot rule out this mechanism of intracellular survival for S. aureus. Although S. aureus is generally classified as an extracellular pathogen, increasing evidence suggests that it has the capacity to infect various mammalian cell lines (Hudson et al., 1995; Lowry, 2000; Hess et al., 2003). It is known that phagocytosed S. aureus can either induce apoptosis of the host cell (Bayles et al., 1998), or survive for several days in the cytoplasm, which is lacking antistaphylococcal activity (Balwit et al., 1994; Proctor et al., 1994). Regardless, the precise mechanisms that allow the survival of epidemic S. aureus strains in A. polyphaga remain to be deciphered.

It should also be noted that although we found that numbers of L. monocytogenes and S. aureus were greater in the presence of A. polyphaga these data do not compare to that obtained using this amoebal strain and L. pneumpohila serogroup 1, subtype Knoxville (Barker et al., 1992). Barker et al. (1992) set up their experiments in exactly the same way as we have in our experiments, except that a MOI of 1 : 1 was used, and they found that bacterial numbers were 2–3 logs higher after 48 h of coculture than in the absence of A. polyphaga.

In conclusion, it is becoming clear that there is a wide spectrum of interactions between pathogenic bacteria and environmental amoebae. Bacterial predation and digestion is at one end of the spectrum and the paradigm of L. pneumophila replication within amoebae is at the opposite end. Indeed increasing research also suggests that between these extremes, there is a variety of interactions where strain dependency is clearly significant and environmental conditions are crucial determinants of the outcome of the interaction. Our data with E. coli and S. enterica serovar Typhimurium highlight the dependency on strain on the outcome of an interaction and we add MSSA, like MRSA (Huws et al., 2006), to the list of pathogens whose survival is enhanced by amoebae.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References

We thank the Lord Dowding Trust for Humane Research (M.R.W.B. and S.A.H.), the UK Department of Health for support (M.R.W.B. and A.W.S.) and also Unilever Research (R.J.M.), who funded this work.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Acknowledgements
  7. References
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