Introduction

Free-living amoebae are ubiquitous and opportunistic protozoa. They can be found in soil, dust, air, seawater, drinking water, swimming pools, sewage, eyewash solutions, contact lenses, dialysis units, and dental treatment units [19, 26]. Acanthamoeba spp. can tolerate a wide range of osmolarity, temperature, salinity, and pH conditions, allowing them to survive in distilled water, tissue cultures, and mammalian body fluids [21]. In recent decades, free-living amoebae have gained increasing attention due to their different roles in the ecosystem. In humans, they cause serious infections such as keratitis and encephalitis, a rare but fatal disease known as granulomatous amoebic encephalitis [22].

Currently, methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen in nosocomial infections, and often may be resistant to several other antibiotics, making multidrug resistance a major public-health problem [23]. Studies have shown that S. aureus can survive or even multiply inside A. polyphaga, which serves as a host or reservoir of various organisms [10].

Some microorganisms have evolved to become resistant to protists, since they are either not internalized or they are able to survive, grow, and exit free-living amoebae after internalization. These “amoeba-resistant microorganisms” include bacteria, viruses, and fungi [9]. These microorganisms can resist to amoebae digestion because of several factors. Some can escape predation and grow in the presence of amoebae, which usually ingest and digest non-resistant microorganisms. Others are able to resist intracellular digestion and survive in amoebic cysts; and still others can not only resist digestion, but also multiply within the trophozoites [24]. Thus, interactions between amoebae and bacteria are complex and depend on the characteristics of both microorganisms, where the result may be beneficial to the amoebae, bacteria, or both [9].

Since amoebae and bacteria occur in many natural and artificial environments, one can assume that they interact in some way. This study investigated the interaction between A. polyphaga and S. aureus (MRSA), through a model of co-culture, and its implications for intrinsic factors of both organisms.

Materials and Methods

Growth Conditions of the Microorganisms

The experiments were performed using a strain of clinical origin of A. polyphaga (ATCC® 30461™) belonging to T4 genotype. The amoebae were grown axenically in PYG medium [0.75% (wt/vol) proteose peptone, 0.75% (wt/vol) yeast extract, and 1.5% (wt/vol) glucose] at 30 °C. The clinical isolate of methicillin-resistant S. aureus (MRSA) was obtained from the secretions of members of a patient from an intensive care unit of a regional hospital (southern Brazil) and was identified by Gram staining technique (Gram-positive), and biochemistry tests of catalase-positive, coagulase-positive, and DNAse-positive. The bacterial isolate was resistant to methicillin. For the experiments, the isolate was grown on agar plate containing Baird Parker (Baird–Parker Agar) at 37 °C for 24 h.

Co-culture Between A. polyphaga and S. aureus (MRSA)

The potential internalization of the S. aureus (MRSA) by amoebae strain was assessed by light microscopy. The A. polyphaga and S. aureus (MRSA) co-cultivation assays were performed at 30 °C in PBS. Briefly, the trophozoites (106 cells mL−1) of A. polyphaga were seeded in a 96-well plates and incubated for 2 h, to allow acclimation and adherence of A. polyphaga to the plate. Then S. aureus (MRSA) (108 cells mL−1) were added to the wells to achieve a multiplicity of infection of 1:100 of amoebae to bacteria, and the plate was incubated for 2, 24, 48, 72, and 96 h at 30 °C to conduct the assessment of cell viability. Experiments were done in triplicate and repeated at least one additional time.

Bacterial Viability After Co-culture

S. aureus (MRSA) were added to a 96-well culture plate with live amoebae (pre-acclimated as described on section above) at a 1:100 of amoebae to bacteria. As control, the bacteria were cultured in PBS without amoebae. The plate was incubated for 24, 48, 72, and 96 h at 30 °C. At each time interval, the cells were resuspended, diluted, and spread on Baird–Parker agar plates, which were incubated at 37 °C for 24 h. After this time, the CFU were quantified. Experiments were done in triplicate and repeated at least one additional time.

Amoebal Viability After Co-culture

The trophozoites were incubated in PYG in a 96-well plate at 30 °C for 2 h prior to treatment. Then the plate was washed twice with PBS and the trophozoites were incubated with S. aureus (MRSA) at a 1:100 ratio. The control was performed by incubating amoebae in PBS without bacteria. At 24, 48, 72, and 96 h, the viability of A. polyphaga was determined by trypan blue exclusion assay [8] and the cells were counted in a Fuchs-Rosenthal counting chamber. Experiments were done in triplicate and repeated at least one additional time.

Amoebic and Bacterial Culture Supernatant and Lysate of Amoebae

To obtain the culture supernatants, trophozoites suspension, in a concentration of 106 cells mL−1, were incubated in 25 cm2 tissue culture flasks at 30 °C for 72 h in PBS [6]. Then the supernatant was collected, centrifuged, and filtrated (0,22 µm). In order to obtain the amoebic lysate, the trophozoites from the culture flasks were prepared in PBS, as described before. The amoebic suspension (106 cells mL−1) was lysed by incubation alternating on ice and at 37 °C for three times, 1 min each [14], followed by seven passages of the suspension through a 27-gage needle. The lysis was assessed by trypan blue assay. After, the lysate and culture supernatant of amoebae were incubated with a suspension of 108 cells mL−1 of S. aureus (MRSA) for 96 h. The bacterial growth was assessed by CFU counts in each incubation period (24, 48, 72 and 96 h).

Encystment of the Amoebae Co-cultured With S. aureus

The trophozoites (106 cells mL−1) were added to 98-well tissue culture plate using a TRIS-buffered medium [13]. After 30 min, amoebae were inoculated with a suspension of S. aureus at 1:1 effector-to-target ratio, and then the plate was incubated at 30 °C allowing encystment of the amoebae for 96 h. As a control, the trophozoites remained alone in an encystment medium. At the end, the number of cysts and trophozoites was determined in a Fuchs-Rosenthal counting chamber. Experiments were done in triplicate and repeated at least one additional time.

Intracellular Presence of S. aureus (MRSA) in A. polyphaga

Fluorescence Microscopy

For the fluorescence microscopy an Olympus BX41 microscope (Olympus America) which has mercury lamp of 100 W, Olympus U-RFL-T brand, with set filter for DAPI Sigma–Aldrich® (4′6′-diamidino-2-phenylindole) having its absorption maximum at a wavelength of 358 nm when exposed to ultraviolet (UV) light, was used. A suspension of the isolated S. aureus (MRSA) was flushed with 100 µL DAPI for about 30–60 min. After this contact time with the dye, the cells were washed with PBS to remove excess DAPI and after, trophozoites of A. polyphaga were added and remained in contact with the stained bacterial suspension for 24, 48, 72, 96, and 192 h.

Confocal Microscopy

For the confocal microscopy, a FV1000 confocal microscopy of Centro de Microscopia e Microanálise (CMM—UFRGS) was used. Fluorescent dye DAPI Sigma–Aldrich® (4′6′-diamidino-2-phenylindole) was used to stain S. aureus (MRSA) having its absorption maximum at a wavelength of 358 nm when exposed to UV light. In addition to this, we used the fluorescent dye Cell Tracker™ CM-DIP (C68H105Cl2N3O)—Thermo Fischer Scientific® with a maximum absorption wavelength of 553/570 nm. The cells of S. aureus (MRSA) and A. polyphaga (in co-cultivation) were incubated in the working solution of the dyes for 5 min at 37 °C and after 15 min at 4 °C. The observation of the intracellular bacteria in the amoebae was given to each incubation interval: 24, 48, 72, 96, and 216 h.

Statistical Analyses

Data are presented as median and were analyzed in 95% of significance (P < 0.05) by Kruskal–Wallis test followed by Dunn’s post-test. For the results obtained in encysting process of A. polyphaga, the Friedman test followed by Dunn’s post-test was used. Statistical analysis and graphs were performed with the Graphpad Prism® 5.0.

Results

Amoebic Viability After Co-culture

The viability of the A. polyphaga after co-culturing with S. aureus (MRSA) was determined using the vital stain trypan blue. Assessments of the co-culture at each incubation time (2, 24, 48, 72, and 96 h) showed that the viability of the amoebae cells decreased (Fig. 1).

Fig. 1
figure 1

Viability of A. polyphaga co-cultivation with S. aureus (MRSA) over time

Full size image

Bacterial Viability After Co-culture

The viability of S. aureus (MRSA) was assessed after co-culture with A. polyphaga, in order to analyze whether contact of the trophozoites with bacteria could affect their survival. This experiment showed an increase in development of the bacterial isolate over the co-culture times (24, 48, 72, and 96 h), quantified by CFU/mL counts. The results were expressed as log10 (Fig. 2).

Fig. 2
figure 2

Viability of S. aureus (MRSA) co-cultivation with A. polyphaga. Bars represent the average of tests carried out in triplicate. Kruskal–Wallis following of Dunn’s post-test. a relative to control. *P < 0.05

Full size image

Amoebic Culture Supernatant and Lysate of Amoebae

To demonstrate whether S. aureus (MRSA) growth in co-culture with A. polyphaga was due to the production of a cellular secretion, the effect of the culture supernatant and the amebic lysate on bacterial growth was evaluated.

The results showed that S. aureus (MRSA) presented better growth when cultivated in the presence of amoeba culture lysate at different incubation times, increasing the amount of CFU/mL. A significant difference was observed between the control and co-culture after 96 h (P < 0.01).

When the effect of amebic culture supernatant on S. aureus (MRSA) growth was tested, a reduction of growth was observed during the incubation period, with a significant difference between control and co-culture after 96 h (P ≤ 0.05, Fig. 3).

Fig. 3
figure 3

Effect of the supernatant of culture and amoebic lysate on bacterial growth. Bars represent the average of tests carried out in triplicate. Kruskal–Wallis following of Dunn’s post-test. a relative to control. *P < 0.05, **P < 0.01

Full size image

Encystment of the Amoebae Co-cultured with S. aureus

Given that the challenge in the management of A. keratitis is the effective eradication of cysts, which are resistant to most antimicrobials, this experiment was conducted to evaluate the effect of S. aureus (MRSA) on encystment of A. polyphaga. Figure 4 shows the percentage of encysted amoebae in relation to the incubation time (24, 48, 72, and 96 h) under different conditions: amoebae + PBS (control); amoebae + MRSA + TRIS; amoebae + TRIS; and amoebae + MRSA (co-culture of both microorganisms).

Fig. 4
figure 4

Influence of S. aureus (MRSA) in process of encystment of A. polyphaga. The number of cysts and trophozoites was determined in triplicate for each condition: amoebae + PBS (control); Amoebae + MRSA + TRIS; Amoebae + TRIS and amoebae + MRSA. Bars represent the average of tests carried out in triplicate. Kruskal–Wallis following of Dunn’s post-test. a relative to 24 h. *P < 0.05, **P < 0.01

Full size image

Significant differences appeared in the cultures under these different conditions, within 24 h of incubation compared to 96 h of incubation. A. polyphaga cultured in PBS (control) showed a value of P < 0.05, the same as when in contact with S. aureus (MRSA) + TRIS (a solution that induces the protozoan to encyst), and when in contact only with bacteria.

A significant difference (P < 0.01) appeared only when A. polyphaga was cultured with TRIS. There was no significant difference between the other incubation times, although the percentage of encystment tended to increase during the incubation period.

The highest percentage of encysted amoebae (approximately 45%) occurred at 96 h of incubation when co-cultured with the isolate of S. aureus (MRSA), with a progressive increase in this percentage with incubation time.

Intracellular Presence of S. aureus (MRSA) in A. polyphaga

When fluorescence microscopy was performed using Sigma-Aldrich® DAPI (Figs. 5, 6), S. aureus (MRSA) was visualized inside trophozoites (Figs. 5, 6) and cysts (Fig. 6) of A. polyphaga at 24, 48, 72, 96, and 192 h of co-culture. It was also observed by means of confocal microscopy with Cell Tracker ™ CM-DIP (Fig. 7) at 24, 48, 72, 96, and 216 h of co-culture, showing that both microorganisms remained viable throughout the experiment. After contact with the dyes, the bacteria were seeded on blood agar plates to test their viability before to put them in touch with amoebae.

Fig. 5
figure 5

Fluorescence microscopy showing S. aureus (MRSA) internalized into trophozoites (a–d) and cyst (e) of A. polyphaga in 24 h (a), 48 h (b), 72 h (c), 96 h (d), and 192 h (e) of co-culture. Bacterial staining performed with DAPI Sigma-Aldrich® (viewed in blue fluorescence). Scale bar 10 µm. (Color figure online)

Full size image
Fig. 6
figure 6

Confocal microscopy showing S. aureus (MRSA) internalized into trophozoites (a–b) and cyst (c–e) of A. polyphaga in 24 h (a), 48 h (b), 72 h (c), 96 h (d), and 216 h (e) of co-culture. Bacterial staining performed with DAPI Sigma-Aldrich® (viewed in blue fluorescence). Scale bar 10 µm. (Color figure online)

Full size image
Fig. 7
figure 7

Confocal microscopy showing S. aureus (MRSA) internalized into trophozoites of A. polyphaga in 24 h (a), 48 h (b), 72 h (c), 96 h (d), and 216 h (e) of co-culture. Bacterial staining performed with Cell Tracker™ CM-DIL (viewed in green fluorescence). Scale bar 10 µm. (Color figure online)

Full size image

Discussion

Interactions of free-living amoebae with a variety of microorganisms (especially bacteria, but also fungi, and some viruses) can benefit the protozoan [9, 12]. The growth and survival of amoebae in the presence of bacteria varies according to the density and species of bacteria [30]. Increased amoebic inviability occurs because the amoebae encyst due to lack of nutrients, showing characteristics such as increased cytoplasmic density, a water expulsion vesicle, and a nucleolus reduced in volume [5].

Interactions between Acanthamoeba and bacteria can either result in a state of protocooperation, or lead to the destruction of one or both of the members [16]. In this study, the amoeba–bacteria interaction destroyed 11% of amoebic cells, which may have been influenced by the production of some substance by S. aureus, thus hampering the development and decreasing the viability of A. polyphaga.

Many bacteria have developed strategies to resist phagocytosis, survive intracellularly, and multiply inside Acanthamoeba species, and are termed amoeba-resisting bacteria or ARB [9]. It has been speculated that some of these strategies have evolved during the long-term coexistence of bacteria and protozoa. These include the formation of extended bacterial cells that are difficult to phagocytose, with more rapid motility, masking and avoiding recognition by the predator, microcolony formation, resistance to phagolysosomes, and intracellular survival and growth or infection [7].

These strategies are seen in numerous bacteria, including Chlamydia, Legionella, Salmonella, Shigella, Pseudomonas, Vibrio, Staphylococcus, and Mycobacterium, which survive in the free-living protozoans and establish a parasitic relationship [15, 27].

Some bacteria, such as Listeria, E. coli, and S. aureus, can survive and multiply in Acanthamoeba spp. through their ability to use and benefit from metabolites secreted by free-living amoebae [7]. Other defense mechanisms that can contribute to resistance in phagocytic cells include the production of bacterial toxins, the presence of protective outer membrane structures, and the reduction of vacuole acidification by bacteria [3].

These results suggest a multiplicity of interactions between A. polyphaga and S. aureus (MRSA), since the amoeba itself may serve as a source of nutrition, and even produce some growth-promoting factor or nutrients, which in turn can be used for growth of the bacterial culture.

The contact between the two microorganisms might have induced the production of compounds by A. polyphaga, which were triggered after phagocytosis of S. aureus (MRSA). Other authors have demonstrated the effect of a culture supernatant of A. castellanii on the growth of L. monocytogenes [10]. These results indicated that the bacteria used compounds secreted by amoeba, which favored their growth. However, the use of amoeba substances is not specific to the genus Listeria.

In a similar study, Hartmanella vermiformis supernatant led to increased survival of Candida albicans, but did not increase the growth of the yeast. Probably the cell debris from the amoebal supernatant could be utilized by the yeast as a nutrient [28].

In the present study, the opposite occurred: the supernatant of the amoeba culture, in some way, interfered with the growth of the S. aureus (MRSA) isolate. This suggests that some compound secreted by the amoeba showed antimicrobial activity against S. aureus (MRSA).

Some bacterial endosymbionts are able to survive inside amoebae cysts, where their intracellular location protects them from adverse environmental conditions [9]. Another study points out that cysts are highly resistant to biocide treatments and are able to survive in adverse conditions, persisting in the stroma during treatment [20]. Therefore, favoring the formation of cysts by S. aureus (MRSA) is quite worrying because it may result in the persistence of the amoeba in the infection site. When Acanthamoeba spp. are subjected to adverse environmental conditions, their phenotype may change, forming cysts. This trophozoite differentiation ability to form cysts is known as a phenotypic change, which may represent the ability of Acanthamoeba spp. to switch protein expression and surface glycoproteins, and in turn can help the amoeba to circumvent the immune system [11].

In this study, the strain of A. polyphaga of clinical origin showed a high percentage of encystment in the presence of S. aureus (MRSA). Probably this organism served as a stressor, causing a phenotypic change in the amoeba.

Thus, it is essential to understand the interactions between amoebae and their endosymbionts, since the virulence may be related to the physiological characteristics of strains of Acanthamoeba [29].

Some studies have shown that the interaction with Acanthamoeba spp. can act as a “training” in which bacteria develop strategies to evade phagocytosis, enhancing their resistance to macrophage attack. The mechanisms used by these bacteria to escape the phagocytic amoebae would be the same used to evade phagocytosis and/or survive in macrophages [12]. In addition, it was observed that S. aureus has the ability to resist phagocytosis and replicate within phagocytes, stimulating production of IL-12 [1]. The use of Acanthamoeba as a bacterial reservoir is an important strategy for these pathogenic bacteria to survive and multiply intracellularly [9]. This concords with the present study, in which S. aureus (MRSA) remained viable for long periods inside A. polyphaga.

The adaptation of bacteria to survive inside free-living amoebae may also contribute to their virulence, making these organisms capable of infecting human cells. The relationship between virulence and replicability within these protozoa was also demonstrated in a study about the growth kinetics of two clinical isolates of L. pneumophila in Acanthamoeba [18].

The ability of E. faecalis and S. aureus grow inside A. polyphaga was also demonstrated. Bacteria were able to persist and grow intracellularly, showing adaptation to the intracellular environment without causing host cell lysis [4].

This study reinforced the hypothesis that Acanthamoeba can play a crucial role in the spread of S. aureus (MRSA) in the community and in the hospital environment.

In addition, the survival and intracellular growth of Acanthamoeba can affect bacteria in some spects: changes in the bacterial phenotype might improve their invasive properties in macrophages, as well as increasing the resistance of the bacteria to extracellular conditions such as antimicrobial agents and disinfectants [2, 25]. Some cell-surface receptors may also affect several bacterial characteristics, including size, motility, phenotypic changes, physiological status, and an array of defense mechanisms that may make the bacteria more or less resistant to predation [17]. In general, amoeba–bacteria interactions are highly complex, and can be influenced by virulence factors of these organisms or by the environmental conditions.

In summary, through the interaction between A. polyphaga and S. aureus (MRSA) in the co-culture model, a small percentage of amoebae became inviable after contact with the bacteria. The results obtained in the present study indicate that S. aureus is an amoeba-resistant microorganism, because the bacteria resisted to the digestion/destruction by the amoebae and used them in several ways to increase their own growth. Moreover, the bacteria stimulated the amoebae to encyst, and probably served as a stressor agent causing this phenotypic change on the amoebae. The mechanisms involved in the interaction between these two organisms merit further study.