Unraveling the interplay between unicellular parasites and bacterial biofilms: Implications for disease persistence and antibiotic resistance

ABSTRACT

Bacterial biofilms have attracted significant attention due to their involvement in persistent infections, food and water contamination, and infrastructure corrosion. This review delves into the intricate interactions between bacterial biofilms and unicellular parasites, shedding light on their impact on biofilm formation, structure, and function. Unicellular parasites, including protozoa, influence bacterial biofilms through grazing activities, leading to adaptive changes in bacterial communities. Moreover, parasites like Leishmania and Giardia can shape biofilm composition in a grazing independent manner, potentially influencing disease outcomes. Biofilms, acting as reservoirs, enable the survival of protozoan parasites against environmental stressors and antimicrobial agents. Furthermore, these biofilms may influence parasite virulence and stress responses, posing challenges in disease treatment. Interactions between unicellular parasites and fungal-containing biofilms is also discussed, hinting at complex microbial relationships in various ecosystems. Understanding these interactions offers insights into disease mechanisms and antibiotic resistance dissemination, paving the way for innovative therapeutic strategies and ecosystem-level implications.

KEYWORDS:

• Bacterial biofilms
• unicellular parasite
• extracellular polymeric substances (EPS)
• nutrients
• biofilm components
• biofilm degradation

Introduction

According to the most recent definition, bacterial biofilms are complex structures formed by microorganism aggregates, either adhering to a surface or existing freely. These microorganisms secrete an extracellular matrix composed of extracellular polymeric substances (EPS), including polysaccharides, proteins, and DNA [1]. Biofilms can be found on a range of natural and artificial surfaces, including medical devices and water pipes, and even within the human body [2]. Biofilms have received significant attention in recent years due to their role in a variety of important medical and environmental contexts, including the persistence of infections [3], the contamination of food [4] and water sources [5], and the corrosion of infrastructure [6,7]. In the medical context, biofilms facilitate the persistence of infections by providing a sheltered environment for bacteria, allowing them to grow, evade the immune system, and resist antimicrobial treatments [8–11] leading to chronic infections [12]. In the human body, biofilms are widespread in various organs and tissues, including the middle ear, upper respiratory tract, oral cavity, cardiovascular system, lungs, stomach, colon, urogenital system, bones, and soft tissue wounds [13]. The presence of biofilms in these organs is often associated with infection [13]. The presence of biofilms is also associated with specific health conditions, such as colorectal cancer, gut wounds, and inflammatory bowel diseases [14]. The presence of biofilms in healthy individuals is more debated. It has been reported for example in the female reproductive tract where aggregates of commensal Lactobacillus species have been observed [15]. Debates surrounding the presence of biofilms in the gut of healthy individuals have been ongoing. Recent advancements in preserving glycocalyx structures within these biofilms during the biological sample fixation process have enabled direct observation, providing substantial evidence confirming their presence within the normal gut [16,17]. One aspect of bacterial biofilms that has received relatively little attention is their interactions with unicellular parasites. Unicellular parasites are single-celled organisms that rely on host cells for their survival and reproduction. They can cause a wide range of diseases in humans [18], animals [19], and plants [20].

Parasite and biofilm can meet in a variety of places, like wastewater, and there is evidences to suggest that bacterial biofilms can act as reservoirs for unicellular parasites [21,22]. The interaction between bacterial biofilms and unicellular parasites may have significant implications for the persistence and transmission of these parasites. Biofilms may provide a protective environment for the parasites, allowing them to evade host immune responses and survive in the presence of antimicrobial agents [23,24]. In this review, we will explore the structural and physiological features of bacterial biofilms, as well as their interactions with important unicellular parasites and the impact of these interactions on both parties involved.

Bacterial biofilm components as a source of nutrients for unicellular parasites

The structure of bacterial biofilms is highly organized, with a distinct architecture that consists of different layers and compartments. At the core of the biofilm is a layer of cells that are embedded in the extracellular matrix, surrounded by a layer of water. The outermost layer of the biofilm is composed of a diverse population of microorganisms, including bacteria, archaea, protozoa, fungi, and algae [25]. One of the key features of bacterial biofilm structure is the presence of channels and pores, which facilitate the exchange of nutrients, waste, and signalling molecules between the biofilm and the surrounding environment [26].

The structural organization of bacterial biofilms plays a crucial role in their ability to adapt and flourish in various environments. The extracellular matrix, which serves as a protective barrier against environmental stressors such as desiccation, Ultraviolet radiation, and antimicrobial agents, also facilitates the formation of complex microbial communities that can carry out important functions such as nutrient cycling and waste degradation [27].

While biofilms are commonly seen as a defence mechanism against protozoan grazing, specific free-living amoebae (FLA) like Acanthamoeba castellanii, which can lead to severe keratitis in healthy individuals and cause amoebic encephalitis, disseminated disease, or skin lesions in immunocompromised individuals [22] and the human parasite Entamoeba histolytica, responsible for amoebiasis [28]. They possess the capability to graze on complex biofilms [29]. The active anti-biofilm component(s) of Acanthamoeba spp. are partially heat-labile, indicating the potential presence of at least one enzymatic activity specifically targeting the biofilm [30]. One possible candidate is alginate lyase, an enzyme that catalyzes the degradation of alginate by β-elimination mechanism, that may provide access to bacteria within biofilms by breaking down the biofilm matrix [31]. Another potential candidate is cysteine proteases (CPs), which are enzymes that break down proteins. These proteases are found in Acanthamoeba spp [32,33]. and have been identified as essential for degrading biofilms in E. histolytica through the degradation of extracellular proteinous matrix components, such as TasA in B. subtilis biofilm [34]. Interestingly, these CPs have been recognized both in Acanthamoeba [33] and in E.histolytica [35–37] as central virulence factors involved in tissue invasion. The release of small-sized secondary metabolites with anti-biofilm properties by Acanthamoeba spp [38]. could explain why certain anti-biofilm activities within this organism show partial resistance to heating [30]. However, the exact nature of these anti-biofilm metabolites still remains to be discovered.

The EPS matrix, constituting almost 90% of biofilm structure, primarily consists of polysaccharides produced and released by bacteria. These polysaccharides serve as scaffolds for cell attachment and can form channels and pores within the biofilm. Consequently, polysaccharides are vital for the structural organization of the biofilm, enhancing its resilience and resistance to environmental stresses [27,39]. Research on glycoside hydrolases (GH) enzymes for degrading polysaccharides in biofilms, leading to their dispersion, has predominantly focused on bacterial GH [40,41]. For example, P. aeruginosa secretes PelA h, a potent GH enzyme capable of degrading Pel, one of the three key polysaccharides constituting the extracellular matrix of these biofilms [42]. Biofilm-degrading enzymes are often produced by bacteria that reside within multi-species biofilms, where they compete with one another for resources. For example, B.subtilis secretes GH like α-amylase or levanase SacC [43,44] to inhibit P.aeruginosa biofilm formation. Yet, the potential of GHs from unicellular predators of biofilms has not been thoroughly investigated. For example, the genome of E. histolytica contains numerous GHs that could be potential candidates for biofilm dispersion (Table 1). However, only a small fraction of these GHs have been extensively studied. Among them, β-amylase stands out as the most researched GHs, proven to be crucial in breaking down mucus and facilitating the parasite’s tissue invasion ability [51]. Another promising source of potent GHs with anti-biofilm activity lies within the protozoa residing in the rumen. These protozoa utilize these enzymes to break down the complex carbohydrates found in the plant material consumed by ruminants [52].

Table 1. A list of E. histolytica GH proteins that exhibit homology with enzymes known to degrade biofilms.

EHI	Protein	homology with	Biofilm destroyed	E value	Identity	Gaps	Ref
EHI_152880	α-amylase	α-amylase of B.subtilis	Staphylococcus aureus, Vibrio cholerae, Staphylococcus aureus and Pseudomonas. aeruginosa	1.00E–16	26%	21%	[45–47]
EHI_130690				3.00E–06	24%	16%	[45–47]
EHI_153100		α-amylase of B. subtilis B1U/1	Pseudomonas.aeruginosa and Staphylococcus aureus	2.00E–06	27%	19%	[48]
EHI_153590	β-amylase	α-amylase of B. subtilis B1U/2	Pseudomonas. aeruginosa and Staphylococcus aureus	1.00E–04	26%	19%	[48]
EHI_023430	Glycosyl hydrolase	α-xylosidase from Aspergillus nidulans	Staphylococcus biofilms	1.00E–39	23%	14%	[49]
EHI_052770				4.00E–31	24%	10%	[49]
EHI_012010	beta-N-acetylhexosaminidase (dispersin B)	Aggregatibacter actinomycetemcomitans	Staphylococcus epidermidis	1.00E–12	29%	10%	[50]

E-value (Expectation value) serves to estimate the probability of encountering random sequence matches during a database search. A lower E-value indicates a more statistically significant similarity. The identity shows the percentage of identical base pairs between the sequence of E. histolytica and that of the reference specimen. The Gap refers to a space inserted in the alignment of two sequences to optimize the alignment score.

Bacterial biofilms contain a variety of proteins, including enzymes, structural proteins, and signalling molecules. These proteins can be involved in the synthesis and modification of the extracellular matrix, as well as in the regulation of biofilm development and behaviour. As previously stated, CPs produced by E. histolytica play a crucial role in breaking down biofilms formed by B. subtilis and E. coli [34]. While the degradation of TasA by E. histolytica CPs explains the mechanism behind B. subtilis biofilm degradation, the situation is different for E. coli curli. Curli, the primary proteinaceous component of enteric biofilms, is resistant to degradation by these CPs [34]. This raises questions about which specific component of the E. coli biofilm is targeted by E.histolytica CPs. Under conventional axenic culture conditions, only a subset of the 35 papain-like ehcp genes present in the E. histolytica genome exhibits high expression levels, specifically ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7 [53]. The degradation of B.subtilis by E.histolytica is marked by the upregulation of specific EhCPs EHI_200690 (ehcp-a14), EHI_117650 (ehcp-a7), EHI_126170 (ehcp-a15) and EHI_151440. It is intriguing to investigate whether this response is specific to B. subtilis biofilm or if it represents a general reaction to bacterial biofilms. Moreover, within the gut environment, mono-species biofilms are more of an exception than a rule [54]. Therefore, understanding the expression profile of these CPs becomes crucial, especially when the parasite is exposed to multi-species biofilms

Giardia lamblia is a unicellular parasite that causes the diarrhoeal illness known as giardiasis [55]. This parasite secretes CPs which disrupt the microbial population of the gut biofilm [56]. Similarly, CPs have been identified in many protozoans known to feed on bacterial biofilms, including Tetrain produced by Tetrahymena [57], a ciliate protozoan commonly found in freshwater environments and as mentioned above, Acanthamoeba [32]. However, the precise role of these CPs in the degradation of biofilms by these protozoans is still unknown.

Another important component of the bacterial biofilm matrix that can be targeted by protozoan enzymes is DNA. DNA plays a critical role in bacterial biofilms by providing structural stability and promoting cohesion within the biofilm matrix [58]. Bacterial DNA is released into the extracellular environment by bacterial that undergo programmed cell death or are killed by antibiotics, chemical and physical stressor, challenging environmental conditions or even predators [59,60]. The released DNA can be used by bacteria to form EPS, which are an essential component of the biofilm matrix [61]. These EPS, in turn, contribute to the structural stability of the biofilm by forming a network of polymers that help to anchor the bacterial cells to each other and to the surface they colonize. Thus, the presence of DNA in bacterial biofilms is critical to the formation, maintenance, and function of these complex microbial communities. The degradation of DNA in bacterial biofilm matrix by DNase may disrupt the structure of the biofilm and affect its function. For example, DNase I degrades the extracellular DNA (e-DNA) present in the P. aeruginosa [62] or Staphylococcus epidermidis [63] matrix rendering the matrix weak and susceptible to antimicrobials. As an additional example, DNase I-coated nanoparticles filled with ciprofloxacin can significantly reduce established P. aeruginosa biofilms by up to 95% and eliminate more than 99.8% of the biofilm. The DNase I coating degrades the extracellular DNA of the biofilm matrix, enabling the antibiotic to penetrate the biofilm [64]. The combination of DNases and amphotericin B has been proposed as a treatment against C. albicans fungus biofilm. Studies have shown that this combination is more effective against C. albicans biofilms than administering the two treatments separately [65]. Protozoa, such as A. castellanii and T. thermophyla, are known to feed on biofilms and produce efficient DNase. As a result, it is possible that DNase produced by these protozoa could play a significant role in biofilm destruction, even though this possibility has not yet been explored [66,67]. Studies have shown that E. histolytica produces DNase, which could help it resist the harmful effects of human extracellular trap (NET) networks [68]. This same DNase activity may also be responsible for breaking down the eDNA present in biofilms. Given the potential of DNase enzymes produced by protozoa to disrupt biofilms, these enzymes could serve as a promising avenue for developing novel therapeutic strategies to combat infections associated with biofilms.

The extracellular matrix of bacterial biofilms contains lipids and biosurfactants, which can make up a substantial proportion of the matrix, such as up to 33% in V. cholerae. These biosurfactants, produced by microorganisms at the air-water interface, affect the exchange of gas and the tension of surfaces, and are especially important for bacterial attachment to and dispersal from oil droplets. For example, the extracellular matrix of P. aeruginosa contains rhamnolipids that are produced through rhamnosyltransferase-mediated biosynthesis [69]. Lipase enzymes are crucial for the metabolism and virulence of unicellular parasites. These enzymes break down triglycerides into free fatty acids and glycerol, which are important sources of energy for the parasites. Studies have demonstrated that lipase enzymes in parasites such as Plasmodium falciparum [70] play an essential role in various cellular processes such as membrane remodelling, lipid metabolism, and virulence. In the case of E. histolytica, the lipase enzymes are involved in the degradation of host cell membranes, allowing the parasite to invade the host tissues and cause tissue damage. Additionally, these enzymes help the parasites to acquire and metabolize host lipids, which are critical for its survival and virulence [71–73]. Lipase from fungal origin have been considered as antibiofilm compounds [74,75]. However, the potential of lipase from protozoan as antibiofilm compounds has not been investigated yet.

Interactions of unicellular parasites: shaping bacterial biofilm composition and functions

Unicellular parasites can impact the formation of bacterial biofilms in various ways, including grazing on bacterial cells, thereby altering the population dynamics within a multispecies bacterial biofilm community [76]. A significant outcome of this grazing activity is the adaptation and development of defensive traits among the bacteria living in the biofilm. For instance, bacteria like Vibrio cholera, Pseudomonas aeruginosa, and Legionella pneumophila, which respectively cause enteric disease [77], opportunistic infections (dermatitis, bacteraemia, and infections of the respiratory and urinary tracts and of other vital organs) [78], and pneumonia [79], can adjust to their protozoan predators. This adaptation leads to the selection of virulence-associated traits, enhancing their survival within the predatory host environment [80].

The impact of unicellular parasites on shaping bacterial biofilm composition and functions has been documented, even in the absence of grazing activity. Leishmania parasites are transmitted to mammal or humans through sandflies bite, resulting in various clinical forms ranging from skin sores to potentially fatal visceral organ damage [81]. Leishmania parasites, which multiply and develop within the host’s reticulo-endothelial system, do not engage in grazing activities. However, in cases involving an endemic strain of Leishmania donovani in Sri Lanka, resulting in cutaneous leishmaniasis instead of visceral leishmaniasis, unique biofilm-positive lesions dominated by Pseudomonas, class Bacilli, and Enterobacteriaceae have been identified. The precise mechanism through which the parasite influences this bacterial community is not yet understood [82]. Another example of a parasite shaping bacterial biofilm composition is Giardia that induce changes in the structure and composition of biofilms within the human intestinal microbiota. Subsequently, bacteria originating from these imbalanced microbiota have the potential to cause epithelial and intestinal abnormalities after the elimination of the enteropathogen. The activity of Giardia CPs has been implicated in this phenomenon [56]. The interaction between trophozoites and biofilm has additional notable implications, especially regarding biofilm antibiotic tolerance. For instance, E. histolytica CPs enhance the sensitivity of the biofilm to antimicrobial agents such as sodium hypochlorite and ampicillin by damaging the biofilm matrix [34].

Biofilm as a source of persistence to protozoan parasites

Biofilms not only provide a favourable environment for the growth and spread of microorganisms but also act as a source of persistence for protozoan parasites like Cryptosporidium parvum oocysts and Giardia cysts that can survive chlorine present in drinking water by being entrapped in pipe wall biofilms [83]. Moreover, biofilms present in wastewater serve as a refuge and a source of nutrients for FLA such as Acanthamoeba sp., Naegleria fowleri, and Balamuthia mandrillaris, which can cause severe infections at the ocular or cerebral level [84,85]. The diversity of FLA in wastewater is influenced by factors such as water temperature, position in the water tank, and other factors, and the biofilm critically affects FLA diversity within the wastewater [86]. These FLA can host human pathogens, such as Legionellaceae, Mycobacteriaceae, and Enterobacteriaceae [87] enhancing the threat of their presence within biofilms. Interestingly, biofilms have the ability to induce the differentiation of amoebae into their environmentally resistant form, known as cysts. This phenomenon is exemplified by Acanthamoeba polyphaga, which forms cysts within 5–7 days of being in contact with a biofilm [88]. To prevent the risks of infections caused by pathogenic FLA and other microorganisms, effective control measures must be put in place to limit the formation of biofilms and control their spread.

Biofilms and their possible influence on the parasites virulence, stress and drug resistance

The EPS found in biofilms, such as alginate produced by Pseudomonas aeruginosa, interferes with the immune system’s defence mechanisms, affecting processes like chemotaxis, complement activation, hypochlorite scavenging, and phagocytosis by macrophages and neutrophils [89]. Furthermore, established biofilms have been observed to induce necrosis in neutrophil, hinder migration, conceal bacteria from detection, and reduce IL-8 production [90]. Considering these findings, it is plausible to speculate that a parasite residing in contact with a biofilm could exploit these properties to evade the host’s immune response.

Biofilms are highly resistant to environmental stress due to their complex and protective matrix, which shields the embedded microorganisms from adverse conditions and external threats [27]. These protective properties can be conferred to parasites leaving inside biofilms. For instance, E. histolytica is shielded against oxidative stress (OS) when it resides inside a B. subtilis biofilm [34]. While the protective mechanism is thought to be related to the presence of an oxygen gradient within the biofilm [40,41], it’s also plausible that certain antioxidant molecules produced by the biofilm, such as Pulcherrimin, an iron-binding reddish pigment [91], could contribute to this protective effect.

Metronidazole, the primary drug used for treating amebiasis [92], experiences reduced efficacy against bacteria in biofilm form. For example, biofilms of Gardnerella vaginalis, a causative agent of bacterial vaginosis, and Porphyromonas gingivalis, involved in periodontitis, show decreased susceptibility to metronidazole compared to their planktonic counterparts [93,94]. One potential mechanism for the resistance of G. vaginalis biofilm to metronidazole is a decrease in metabolic activity during antibiotic exposure, thereby preventing drug reduction and consequent activation [95]. Considering that many redox enzymes have their expression downregulated in E. histolytica present inside biofilms [34], it is tempting to hypothesize that reduced activation of metronidazole occurs in the parasite embedded within the biofilm, thereby diminishing its effectiveness.

Interaction between unicellular parasites and fungal-containing biofilms

While this review predominantly focuses on bacteria-based biofilms, it’s essential to note that many medically important fungi also produce biofilms, including Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, and Pneumocystis [96]. A recent review has documented some of the limited known interactions between amoebas and fungi [97]. For instance, it has been reported that the culture supernatant of the free living amoeba Vermamoeba vermiformis and A. castellanii provides Candida auris promotes the yeast survival and proliferation [98]. Similar observations have been made with Aspergillus fumigatus, a prevalent mould, within a biofilm structure, having its growth promoted by V. vermiformis [99]. The presence of C. albicans in the human gastrointestinal tract raises the possibility of interactions with enteric parasites. Preliminary findings from our laboratory show that E. histolytica can phagocytize C. albicans hyphae, supporting this concept. The importance of this lies in the fact that the parasite’s ability to phagocytize the hyphal form of C. albicans is noteworthy, particularly as Dictyostelium discoideum, a soil amoeba, are poorly efficient in engulfing the hyphal form of C. albicans [100]. The most likely relationship between E. histolytica and C. albicans is a predator-prey interaction. However, it is also conceivable that the fungus benefits from amoeba metabolites for its growth, or the amoeba may influence the pathogenicity of the fungus, as it has been proposed in other amoeba-fungi systems [97]. In summary, although our preliminary data provides insights into the interaction between unicellular parasites and fungal-containing biofilms, it is crucial to recognize the current lack of comprehensive research in this specific domain and emphasize the necessity for further exploration into these interactions.

Conclusions

Recent research has highlighted the significant influence of unicellular parasites on the formation, structure, and properties of bacterial biofilms (Figure 1). These findings have implications for understanding the role of parasites in diseases and the spread of antibiotic resistance genes within microbial communities. Further research is needed to fully understand the mechanisms underlying these interactions and to develop effective control strategies to prevent waterborne and foodborne transmission outbreaks. Promising avenues for future investigation include the use of enzymes from unicellular parasites to disrupt bacterial biofilms and the development of new antimicrobial therapies based on the mechanisms by which unicellular parasites feed on and disrupt biofilms. Additionally, understanding the interactions between unicellular parasites and bacterial biofilms in the context of the larger microbial ecosystem could have implications for ecosystem-level processes such as nutrient cycling and waste degradation. Overall, continued research in this field has the potential to lead to important advances in the treatment and prevention of biofilm-associated infections.

Figure 1. Summary of the review. this review emphasizes three key aspects of the relationship between unicellular organisms and biofilms. Firstly, various unicellular organisms exhibit the ability to graze and degrade biofilms through potent enzymatic activity such as CPs [34] and GH (as indicated in Table 1). Secondly, parasites influence the formation and composition of biofilms, leading to dysbiosis. Lastly, biofilms serve as shelters for unicellular organisms residing within them, offering protection against different stresses like environmental factors, drugs, and immune attacks. This protective environment potentially influences the virulence and life cycle of these organisms.

Author contributions

All authors have read and agreed to the published version of the manuscript.

Acknowledgements

We are thankful to Dr. Kornitzer, the Faculty of Medicine at Technion, for generously providing the C. albicans strain used in our preliminary experiment investigating the interaction with E. histolytica.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data Availability statement

NR.

Additional information

Funding

The work was supported by the Israel Science Foundation (3208/19) and the Ministry of Science and Technology, Israel (1020546).