- Short report
- Open Access
Acanthamoeba and bacteria produce antimicrobials to target their counterpart
© Iqbal et al.; licensee BioMed Central Ltd. 2014
Received: 30 October 2013
Accepted: 28 January 2014
Published: 30 January 2014
In the microbial ecosystem, microbes compete for space and nutrients. Consequently, some have developed the ability to kill or inhibit the growth of other competing microbes by producing antimicrobial substances. As the ‘producer’ species are generally immune to these substances, their compounds act on the competing microbial species and give the producer more space and access to nutrients for growth. Many currently used antibiotics were developed by exploiting this potential of certain microbes.
Here, the free-living amoeba, Acanthamoeba castellanii, was investigated for its antibacterial activity against representative Gram positive and Gram negative bacteria, while bacterial isolates were tested for their anti-amoebic properties. Conditioned medium from A. castellanii showed remarkable bactericidal properties against methicillin-resistant Staphylococcus aureus (MRSA) exhibiting almost 100% kill rate, but had limited effect against Acinetobacter sp., Pseudomonas aeruginosa and vancomycin-resistant Enterococcus faecalis (VRE). Similarly, the conditioned medium of E. coli K1 and Enterobacter sp., exhibited potent anti-Acanthamoebic effects in a concentration-dependent manner. Conditioned media of Acanthamoeba, E. coli K1 and Enterobacter sp. showed no cytotoxicity in vitro when tested against human brain microvascular endothelial cells. Active molecule/s in aforementioned amoebic and two bacterial conditioned media were 5 – 10 kDa, and <5 kDa respectively.
A. castellanii conditioned medium showed potent bactericidal properties against MRSA. The active molecule(s) are heat- and pronase-resistant, and in the 5 to 10 kDa molecular mass range. Contrary to this, E. coli K1 and Enterobacter sp., conditioned medium showed anti-amoebic effects that are <5 kDa in molecular mass, suggestive of active metabolites.
Infectious diseases account for 68% of global child mortality, with the vast majority occurring in developing countries [1–3]. Novel antimicrobial agents are urgently needed to counter multi-drug resistant pathogens . Most clinically used antibiotics are either derived from natural products especially from microbes or semisynthetic derivatives of these molecules [5–7]. In parallel, there is a need to expand the range of organisms that can be tapped in our search to discover natural product antibiotics. Given the importance of small molecules to microbial ecology, e.g., penicillin, which is produced by Penicillium as a defence mechanism [8, 9], it is likely that other microbes can also contribute to the discovery of new antibiotics. Acanthamoeba is one of the most ubiquitous protists that has been isolated from diverse environments . In its natural habitat, i.e., soil and water, Acanthamoeba feeds on different bacteria and plays an important role in the regulation of bacterial populations . However, certain bacteria especially pathogenic bacteria have developed strategies to escape the killing mechanisms of Acanthamoeba and instead use the amoeba as a Trojan horse or reservoir for their own benefit [12, 13]. Some bacteria protect themselves from preying amoeba by secreting soluble anti-Acanthamoeba substances in their medium [14–16] or directly injecting such substances into Acanthamoeba through the type III secretory system . These findings suggest that Acanthamoeba and Bacteria encounter each other in the environment routinely. But, how Acanthamoeba survive the onslaught of overwhelming bacterial population remains incompletely understood. Thus, it is reasonable to hypothesize that, in addition to bacteria, Acanthamoeba also possesses antibiotics to counter bacterial attack. As Acanthamoeba and bacteria thwart each other in their natural habitat, the overall aim of this study was to determine the anti-bacterial activities of Acanthamoeba and also the anti-Acanthamoebic properties of selected bacterial strains.
A keratitis isolate of Acanthamoeba castellanii was obtained from the American Type Culture Collection (50492) and grown axenically in PYG medium [proteose peptone 0.75% (w/v), yeast extracts 0.75% (w/v) and glucose 1.5% (w/v)] in tissue culture flasks aerobically at 30°C without shaking [17–19]. A. castellanii conditioned media (ACM) were prepared by incubating confluent cultures (~2×106 trophozoites/mL) for 48 h in PYG. Given confluent cultures, amoebae number increased slightly to ~3×106 trophozoites/mL. Next, cell-free medium was collected by centrifugation (1000 × g for 5 min) and filtered using 0.22 μM pore size filters. In some experiments, the ACM was treated at 95°C for 10 min to inactivate A. castellanii extracellular proteases.
Bacterial isolates used in the present study were Acinetobacter sp., Aeromonas hydrophila, Enterobacter aerogenes, Enterobacter sp., Escherichia coli K1, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Shigella flexneri, and Vancomycin-resistant Enterococcus faecalis (VRE). Escherichia coli K1 strain RS218 (O18:K1:H7) and MRSA have been described previously [20, 21]. They were isolated from the cerebrospinal fluid of a meningitis patient  and blood culture of septicemia patient , respectively. Enterobacter sp. was isolated as a contaminant in Acanthamoeba culture, and all other bacteria are clinical isolates obtained from the Aga Khan University Hospital, Karachi (available upon request). Bacterial conditioned media (BCM) were prepared by culturing single colonies (grown on nutrient agar plates) in 100 mL PYG medium and incubating for 24 h at 37°C aerobically, with shaking. These conditions resulted in growth of all bacterial cultures to stationary phase of their growth. The bacterial cultures were centrifuged at 4000 × g at 4°C for 20 min. The supernatants were collected and filter sterilized using 0.22 μM pore size membrane filters. In some experiments, BCM were treated at 95°C for 10 min to inactivate extracellular enzymes.
Bacterial conditioned media inhibited A. castellanii growth
Enterobacter sp. and E. coli K1 conditioned media produced A. castellanii death
For amoebicidal activity of BCM, A. castellanii trophozoites (106 amoebae/0.5 mL/well) were incubated in PYG with different amounts of BCM in 24 well plates at 30°C for 48 h. Following this incubation, amoebae viability was determined by adding 0.1% Trypan blue and number of live (non-stained) and dead (stained) A. castellanii were enumerated using a hemocytometer. At 50% concentration, E. coli K1 and Enterobacter sp. BCM did not affect A. castellanii viability (0% and 0.7% respectively), while at 100% concentration, BCM of both E. coli K1 and Enterobacter sp. exhibited more than 90% amoebicidal effects (Figure 1C).
Anti-Acanthamoebic compound(s) in Enterobacter sp. and E. coli K1 conditioned media are pronase-resistant and <5 kDa in molecular mass
Enterobacter sp. and E. coli K1 conditioned media are not cytotoxic to primary human brain microvascular endothelial cells
Cytotoxicity assays were performed as previously described . Briefly, assays were performed in 24 well plates containing confluent human brain microvascular endothelial cells (HBMEC) monolayers. The conditioned media (250 μL) or PYG medium (250 μL) were added in 250 μL RPMI-1640. Plates were incubated at 37°C in a 5% CO2 incubator and monitored for monolayer disruptions over the period of 24 h. After this incubation, the supernatants were collected from each well, centrifuged to remove cellular debris and then cytotoxic effects were determined by estimating the amount of lactate dehydrogenase release from HBMEC using a Cytotoxicity Detection kit (Roche Applied Sciences). The percent cytotoxicity was calculated as follows: % cytotoxicity = (sample value – control value) / (total LDH release – control value) × 100. Control values were determined by incubating HBMEC monolayers with RPMI 1640 alone and total LDH release was obtained by completely lysing the HBMEC using 1% Triton X-100. Cytotoxicity assays were performed to determine the effects of the Enterobacter sp., and E. coli K1 BCM on primary HBMEC. Neither E. coli K1 BMC nor Enterobacter sp. BCM produced host cell cytotoxicity (0.3% and 2% host cell death respectively). As BCM was prepared using PYG, similar volume of PYG alone (250 μL) were used as controls. The results revealed that PYG alone exhibited neither monolayer disruptions nor HBMEC cytotoxicity (ca. 2%).
A. castellanii conditioned media exhibited potent antibacterial activities against MRSA
Acanthamoeba conditioned medium (ACM) exhibited selective antibacterial properties
Bacteria + Acanthamoeba conditioned medium (ACM)
CFU remaininga(Bacteria + ACM)
Bacterial kill (%)
Methicillin-resistant Staphylococcus aureus (MRSA, 10 μL) + ACM (190 μL)
Acinetobacter (10 μL) + ACM (190 μL)
1 × 1010
Pseudomonas aeruginosa (10 μL) + ACM (190 μL)
6 × 109
Vancomycin resistant Enterococcus faecalis (VRE, 10 μL) + ACM (190 μL)
3.75 × 105
8.12 ± 9.8
MRSA (10 μL) + heated ACM (190 μL)
MRSA (10 μL) + heated ACM (180 μL) + PYG (10 μL)
6.65 × 104
93.3 ± 9.9
MRSA (10 μL) + heated ACM (140 μL) + PYG (50 μL)
1.55 × 107
MRSA (10 μL) + heated ACM eluate of 10 kDa spin exclusion column (190 μL)
MRSA (10 μL) + heated ACM eluatee of 5 kDa spin exclusion column (190 μL)
1.34 × 1010
MRSA (10 μL) + heated-pronase-treated ACM (190 μL)
The presence of antimicrobials in microbes is not a novel concept and they are known to be an ancient weapon in the defense against the growth of other microbes. Since the discovery of penicillin produced in nature by mould, Penicillium in 1928 , a large number of microbes have been tested for the isolation of potentially useful antimicrobials, while there has been renewed interest in finding new antibiotics from unique sources such as actinomycetes, cyanobacteria and unculturable bacteria, in addition to known sources such as streptomycetes.
The ability of bacteria to thrive at high population densities and the predatory role of Acanthamoeba in the control of bacterial populations in the environment as well as the ability of the amoeba to act as a “Trojan horse” of the microbial world suggests that bacteria-protists are involved in convoluted interactions [11–16]. The precise nature of such complex interactions is not clear, but it is shown here that both bacteria and amoebae are able to counter-attack each other and the overwhelming microbial densities by secreting/harboring antimicrobials. Among four bacteria tested, it is interesting that ACM exhibited selective and potent activity against MRSA. Superbugs, such as MRSA have shown the ability to cause untreatable infections and have become a major threat in our fight against bacterial infections . The selective and potent nature of ACM against Gram-positive MRSA and partly against VRE, but not against Gram-negative P. aeruginosa is interesting. It is possible that P. aeruginosa and Acanthamoeba are involved in complex interactions where the bacteria can employ strategies to defend itself and counter amoebic attack . Future studies will test ACM against a large panel of diverse bacterial pathogens to determine broad-spectrum or species-specific antibacterial properties. The discovery of antibacterial compound(s) from Acanthamoeba will open avenues for future studies in other free living amoebae/protists as a potential source of antibiotics for the treatment of emerging bacterial pathogens such as MRSA. Because of increasing trends in antibiotic resistance in different pathogenic bacteria , the present findings are promising in our efforts to treat infections caused by drug-resistant bacteria.
Similarly, among various bacterial isolates tested, BCM of Enterobacter sp. and E. coli K1 exhibited measurable amoebistatic and amoebicidal properties. Future studies will address the question of whether these BCM have selective action against Acanthamoeba or have wide scale activities against different protist pathogens. Interestingly, neither ACM nor BCM were cytotoxic to brain endothelial cells in vitro, suggestive of the selective action of these conditioned media against MRSA and Acanthamoeba, respectively. Future studies will further test the specificity and the absence of putative target(s) in human cells using primary cells of various origins. Overall, the present findings are promising in our search to find additional sources of antimicrobials with novel modes of action to confront this menace. Work is currently underway to characterize further the antimicrobial properties of ACM and BCM and active components, possibly novel secondary metabolites in the case of BCM, being resistant to pronase and heat treatment and less than 5 kDa in molecular mass. As antimicrobial resistance is on the increase, finding novel non-toxic antimicrobial compound(s) from a variety of species prevalent in clinical settings is crucial. It is hoped that these molecules will eventually be developed into treatments for bacterial and amoebic infections that are increasingly resistant to currently available drugs, however, this will require intensive research in the next few years.
This work was supported by grants from the Aga Khan University.
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