The Interconnection between Virulence Factors, Biofilm Formation, and Horizontal Gene Transfer in Enterococcus: A Review

Abstract

Bacterial evolution, ecology, and environmental adaptability are all linked processes that facilitate its survival. Enterococci are opportunistic pathogens with an ability to form biofilms during infections and this process is influenced by several virulence factors. The species constitute a substantial group of commensal bacteria and have been found to have a number of virulence factors that are thought to be crucial in aggravating diseases that they cause. These pathogens are essentially resistant to antibiotics and this capability is attributed to their ability to acquire and transfer drug-resistant genes via horizontal gene transfer leading to diverse phenotypes. Enterococci have several virulence factors that confer their resistant nature and they are broadly categorized into two: surface factors that aid with colonization of the host and proteins that are secreted by the pathogen to damage the host tissues. Biofilm formation by enterococci is attributed to its surface components and aggregation substances that aid in the adherence to the host’s surface and hence limiting antibiotic penetration. Bacterial biofilms also contribute to its resistance to antimicrobial drugs and hence posing a challenge in attempts to eradicate the pathogen. Therefore, the interconnection between virulence, biofilm formation, and horizontal gene transfer leads to pathogenesis in enterococci.

1. Introduction

Enterococcus spp. are widespread gram-positive and facultative anaerobic bacteria that live in the human gastrointestinal tract, vaginal tracts, and the oral cavity [1]. The Enterococcus genus is also present in a wide variety of dietary products that are dairy, meat, and vegetable-based [2]. The capacity of these microbes to withstand high salt concentrations (6.5% sodium chloride) as well as a broad range of temperature and pH values (4.4 to 9.6) is one of their main characteristics [3].

Enterococci are one of the leading causes of life-threatening healthcare-related infections. Because of their innate tolerance to a variety of antibiotics, enterococci can quickly develop significant levels of drug resistance through horizontal gene transfer [4]. They commonly cause infections of the urinary tract and wounds in hospitals, as well as endocarditis or bacteremia [5]. Most human enterococcal infections are caused by Enterococcus faecalis, accounting for 80–90% of cases, and E. faecium for most of the remaining cases. A few other enterococcal species that are not often associated with human illness are E. avium, E. casseliflavus, E. durans, E. gallinarum, E. hirae, E. mal-odoratus, E. mundtii, E. raffinosus, and E. solitaries [6].

Modern agriculture’s use of antibiotics has produced a reservoir of resistant enterococci in food animals and animal-derived food [7]. These enterococci are likely to provide virulence-associated genes and resistance to other enterococci that live in pets and humans because these genes seem to spread freely between enterococci from different reservoirs, regardless of their apparent host association [7]. The inherent capacity of enterococci to easily obtain, amass, and disseminate extrachromosomal elements that encode virulence traits or genes resistant to antibiotics confers benefits to their survival in atypical environmental stressors and partially accounts for their growing significance as nosocomial infections. Therefore, because of their capacity to form biofilms, the presence of virulence genes, and their propensity to shelter and transmit virulence to other pathogens through horizontal gene transfer, enterococci have a pathogenic potential that is cause for concern. We will be able to discover how these hardy bacteria have adapted to the particular circumstances of the hospital setting in the antibiotic era by comprehending the forces that have influenced the enterococci’s evolution over years.

2. Virulence in enterococci

Virulence factors are features that define pathogenesis of majority of infections. Adhesion to host tissues is a prerequisite for enterococci to function as pathogens. Enterococci face a very different environment during tissue invasion than they do at colonization sites. This environment includes phagocytic leukocytes, low levels of critical nutrients, increased redox potentials, and other host defense mechanisms [8]. It is probable that enterococci that cause infection express genes that promote proliferation in these different environmental circumstances [3]. Enterococci are known to express components that enable adhesion to extracellular matrix and host cells, promote tissue invasion, influence immunomodulation, and result in harm caused by toxins.

The pathogenesis of the majority of infections is characterized by potential traits known as virulence factors. Although virulence factors have been linked to a pathogenic potential, their primary function is to enhance bacterial fitness. These factors enable resistance to nonspecific defensive mechanisms, colonization, adhesion to the cells of the host, and tissue invasion [9]. The virulence of an organism is controlled by virulence coding genes present on the genome in special regions which are termed pathogenicity islands.

Numerous virulence factors, which can be categorized into two groups, are thought to play a key role in the pathogenesis of enterococci. These groups include cell surface virulence factors such as aggregation substances (asa1), enterococcal surface protein (esp), and collagen-binding protein (ace), as well as secreted virulence factors like hyaluronidase (hyl), cytolysin (cylA), and gelatinase (gelE) [8]. Experiments on animal infections revealed that several of these virulence characteristics could make Enterococcus strains more pathogenic. The death rate rose dramatically in animals infected with bacterial strains exhibiting aggregating material and cytolysin in a rabbit model of E. faecalis endocarditis. Gelatinase-active strains appear to be more pathogenic in mice with peritonitis than strains lacking in the enzyme [10].

2.1 Secreted virulence factors

Enterococcal pathogenicity in virulence models has been linked to a number of secreted factors. In addition to providing an arsenal for attack or defense when other organisms are present, some of these secreted factors have also been linked to the production of toxins, obtaining nutrition from the environment, colonization, and immune evasion [11]. These include:

2.1.1 Cytolysin

This protein is cytolytic and can lyse erythrocytes from humans, horses, and rabbits. It contributes to the pathogenicity of E. faecalis by causing blood hemolysis. It has been demonstrated that enterococci strains that produce hemolysin are virulent in both animal models and human infections, and they are linked to higher infection severity [12]. By inoculating enterococci on recently prepared beef heart infusion agar supplemented with five percent horse blood, hemolysis can be identified. Plates were assessed 24 and 48 hours after being incubated overnight at 37°C in a carbon dioxide atmosphere. A clear zone of β hemolysis surrounding colonies on horse blood agar is considered positive. It was discovered that a unique, two-component regulatory system using a quorum sensing mechanism controls the expression of either cytolysin or hemolysin [2].

Its dual action, exhibiting both bactericidal toward gram-negative bacteria and cytolytic properties for leukocytes, erythrocytes, and macrophages led to its naming. Cytolysin is a plasmid or chromosomally-encoded toxin [13]. It is expressed by the genes cylLL and cylLS. In addition to appearing to have some effect on mouse red and white blood cells, cytolysin also appears to play a role in endocarditis and endophthalmitis [3]. Genes encoding for cytolysin have been discovered in Enterococcus strains isolated from infections as well as in those that comprise the commensal microbiota. Numerous findings show that they are frequently found in strains that have been isolated from dietary sources that come from both plants and animals [2].

2.1.2 Hyaluronidase

Hyaluronidase is primarily a degradative enzyme that operates on hyaluronic acid (hyaluronate, hyaluronan) and is linked to tissue injury as a result of its action. It is widely distributed in nature, appearing in everything from snake venom to mammalian cells like spermatozoa to parasites like leeches and hookworms.

Along with other bacteria, streptococci creates large amounts of it [13]. By depolymerizing connective tissues’ mucopolysaccharide moiety, hyaluronidase promotes bacterial invasiveness. Since the breakdown products of its target substrates are disaccharides that bacteria may transport and metabolize intracellularly, hyaluronidase may play the role of providing nourishment for the bacteria. It is also thought to make bacterial toxins and their transmission through host tissues easier. Hyaluronidase not only has a negative effect on its own but can also exacerbate the negative effects of other bacterial toxins by opening the door for their negative effects [3].

2.1.3 Gelatinase

Gelatinase is a metal-loendopeptidase that is extracellular and dependent on zinc. Its molecular weight is around 30 kDa [2]. This enzyme can hydrolyze various bioactive peptides, such as proteins attached to pheromones, gelatin, collagen, elastin, and hemoglobin. Located on a chromosome, the gelE gene encodes gelatinase.

A major factor in the spread of enterococci within their host is the enzyme gelatinase’s capacity to harm host tissues. Biofilm production also requires gelatinase as it facilitates the first stage of biofilm development or the aggregation of cells in microcolonies [14]. Three genes (fsrA, fsrB, and fsrC) make up locus fsr, which regulates the transmembrane protein FsrB, which in turn controls the gene [2]. The regulatory proteins FsrA, FsrB, the pheromone transporter GBAP, and the histidine kinase FsrC are encoded by the fsrABC gene set. Mutations resulting from deletions in locus fsr result in mutants that lack gelatinase production even though they carry the gelE gene, which lessens their pathogenic potential. It has been demonstrated that fsrA, fsrB, or fsrC gene mutations decrease biofilm synthesis by 28 to 32% [2].

2.1.4 Catalase

One well-known enzyme that exists in each of the three realms of life is catalase. By catalyzing the breakdown of hydrogen peroxide (HP) into water and oxygen, it shields the cell from HP’s oxidative damage. The reactive oxygen species (ROS) in the biosphere is HP [9]. As a byproduct of oxidase activity, photosynthetic electron transport chains, oxygen activation, and respiratory electron transport chains, among other aerobic metabolic processes, it is created. HP is reduced to water in the first phase of the catalase reaction, creating an oxoiron and a cationic heam radical (compound 1 (FeIV⅑O ion)). Water and oxygen are released in the second stage when the reaction of a second HP completes the dismutation process. In the resting FeIII state, the enzyme regenerates. Compound II, an inactive partially oxidized dead-end version of the enzyme, is prevented from building up by NADPH-binding catalases. There are three kinds of catalases: The prokaryotic Mn-catalases, also known as haem catalases, are bifunctional catalase peroxidases that are absent from both plants and mammals and have both catalytic and peroxidative activity. Nine genes have been found to be critical for the expression of catalase activity, dispersed throughout five chromosomal loci excluding katA, which codes for the catalase enzyme protein [9].

2.2 Cell surface virulence factors

A few virulence factors are firmly bonded to the enterococcal surface, in contrast to the secreted factors, which may also be loosely adhered to it [11]. The adherence of these surface-associated factors has been linked to colonization, which is one of the initial stages of a potential infection. They have additionally engaged in immune evasion, which is necessary when a commensal relationship is established but can be harmful to the host in the event of an infection [11]. Elements of the cell surface are crucial for a number of distinct bacterial defense mechanisms, such as the creation of biofilms [15].

2.2.1 Aggregation substance

The bacterial adhesin known as aggregation substance (AS), which is plasmid-encoded and pheromone-responsive, facilitates plasmid transmission by effectively mediating contact between the donor and recipient bacteria. Although the donor cell expresses AS, the recipient cell’s surface must express binding substance (BS), which is a chromosomally encoded cognate ligand for AS, in order for the bacterial conjugation process to occur [13]. Apart from its adhesive role in the bacterial conjugation process, AS mediates E. faecalis adherence to a range of eukaryotic cells in vitro, such as intestinal epithelial cells and renal tubular cells. Aggregation substance is also involved in antibiotic gene transfer. Sex pheromones mediate the transfer of large conjugative plasmids containing a variety of highly similar adhesins, which collectively constitute the aggregation substance. This process is known as facilitated conjugation system [2]. The group of adhesins includes Asp1, Asc10, and Asa1 and are encoded by three distinct conjugative plasmids: pPD1, pCF10, and pAD1, respectively.

2.2.2 Collagen binding protein: Ace

Another surface protein with adhesive qualities is called Ace (Accessory colonization factor) also known as adhesion of collagen, from E. faecalis, and it has a molecular weight of roughly 74 kDa. Ace is the gene that codes for it [2]. The protein was found to be present in E. faecalis strains that were recovered from both healthy carriers and enterococcal infection patients, suggesting that the species may be identified using this characteristic. Ace, like the AS protein, is involved in the process of colonization through binding to extracellular matrix (ECM) proteins, including type I and IV collagen. Ace is a surface protein that belongs to the family known as MSCRAMM (microbial surface component recognizing adhesive matrix molecules). Its high affinity and specificity for binding a ligand are attributed to its LPXTG (L - leucine, P - proline, X - any amino acids, T - threonine, G - glycine) sequence. Other members of the family include Acm (adhesion of collagen, from E. faecium) and Scm (second collagen adhesion, from E. faecium) [3].

2.2.3 Surface protein - Esp

The largest known enterococcal protein is enterococcal surface protein (Esp), which has a molecular weight of roughly 200 kDa. The pathogenicity island (PAI), which also houses proteins in charge of the active outflow of antibiotics, is home to the esp. gene encoding this protein. Horizontal gene transfers between E. faecalis and E. faecium are most likely the cause of this site [9]. Animal models have demonstrated that the surface protein Esp plays a role in the colonization and persistence of E. faecalis during urinary tract infections. Esp has been linked to the facilitation of E. faecalis primary attachment and biofilm formation on abiotic surfaces [13].

2.3 Biofilm formation and its composition

Biofilm is a multiple of cells attached irreversibly on several abiotic and biotic surfaces and enclosed in a hydrated matrix of exopolymeric substances, polysaccharides, proteins, and nucleic acids. Enterococci’s ability to build biofilms is one of its most significant pathogenicity traits [16]. The nutrients in the growing media, including glucose, serum, CO2 and iron availability, osmolarity, pH, and temperature, affect the formation of biofilms in various bacteria. Biofilm formation in a variety of gram-positive bacteria, such as E. faecalis, is regulated by carbohydrate metabolism [17]. The ability of microbes to form biofilms on surfaces is a key virulence factor in that it protects them from external environments and guarantees longevity [18]. Biofilm formation is a result of bacterial cells aggregating together and enclosed in a self-produced extracellular matrix forming a mat-like substance on surfaces that confers protection from the external environment. Biofilms occur almost everywhere in nature ranging from living to nonliving surfaces [17].

Generally, biofilms comprise of bacterial cells, extracellular polymeric substances (EPS), proteins, ribonucleic acid, polysaccharides, and water being the major components. The water channel ensures the biofilm is hydrated and nutrients are available for the microbes [19]. Enterococci bacteria are known for their potential to form highly resistant biofilms which often comprise dense populations of bacterial cells enclosed in a humidified matrix of glycans, proteins, and nucleic acids. These types of biofilms are known to attach to surfaces permanently in specific environmental conditions making them resistant to different antibiotics [19]. Biofilm formation in enterococci contributes to its virulence by conferring its surface adherence ability, a key primary role, and hence the capability to bind to suitable environmental surfaces such as the urinary tract systems and oral fissures. Enterococcal biofilms make them more resilient and aid in their persistence throughout infection as well as environmental and food sector contamination. Its inherent resistance to antibiotics is increased by their capacity to form biofilms, which poses a significant challenge to the treatment of infections [20].

2.3.1 Initial attachment

The first stage in the formation of a biofilm is called bacterial surface attachment, and it involves several lipids and proteases [21]. Before biofilm attachment, a few number of cells use weak van der Waals forces to adhere to surfaces. In vitro and in vivo biofilm attachment is mediated by the biofilm-associated pilus (Ebp). Research has demonstrated that in in vivo models of infectious endocarditis, urinary tract infection (UTI), and catheter-associated UTI (CAUTI), the enzyme beta protein (Ebp) of Enterococcus faecalis plays a role in the early biofilm formation [21]. Adhesins are molecules that eukaryotic mucosal surfaces employ to adhere to digestive commensals (Enterococcus) and facilitate surface colonization. Should Enterococcus fail to properly cling to the surface, stomach peristalsis would likely eradicate the microorganism [22].

2.3.2 Irreversible attachment and microcolony development

After adhering to a surface, microorganisms multiply and divide to form a matrix of cells known as a microcolony. Extracellular polymeric substances (EPS) are produced in greater quantities when microcolonies form on the surface [23]. EPS is crucial for the formation of a biofilm because it keeps cells closely together and facilitates adhesion to the surface. Polysaccharides, lipids, proteins, extracellular DNA, and biopolymers are the constituents of extracellular polymeric substances (EPS). In comparison to mature biofilms, this cell matrix is typically less structurally intact and represents a transitional stage in the development of biofilms [24].

2.3.3 Biofilm maturation

At this stage, the EPS and microcolonies have grown into substantial three-dimensional structures (macrocolonies) in well-established mature biofilms. Typically, at this point, the EPS matrix makes up 90% of the dry mass and the amount of microbes is just 10% of it. The biofilms will have both sparse areas that serve as pathways for the movement of water, nutrients, and oxygen, and dense areas with lots of bacteria in them at this point [23].

2.3.4 Biofilm dispersal

Population density in a biofilm influenced by quorum sensing causes the dispersal of biofilms from one location to another [14]. Afterward, scattered cells may colonize or infect different body parts. Dispersal can be physical, where flow pressures lead to shearing, or active, where cell signals or environmental changes drive it.

In addition to showing improved resistance to immunological reactions and antibiotics, biofilm cells frequently have changed phenotypes in terms of growth rate and gene transcription [23]. The high-density, well-organized, and diversified microbial community in biofilms allows for physical cell-to-cell contact and concentrates on a variety of chemical substances, including extracellular DNA and communication signals. These factors make biofilms ideal for bacterial interactions. Furthermore, multispecies communities are commonly seen in environmental biofilms [23].

2.4 Horizontal gene transfer

The ability of bacteria to transfer genes between related and unrelated species is known as horizontal gene transfer (HGT). This process is crucial for the adaptation of bacterial species to novel environments and stresses like antibiotic pressure. The 1970s saw the beginning of research on transferable antibiotic resistance in enterococci [20]. The genomes of enterococci are remarkably flexible and have been demonstrated to impart resistance features to more pathogenic species such as S. aureus.

Antibiotic-resistant bacteria persist and spread in clinical settings, livestock breeding, and soil due to the horizontal transfer of resistance genes and antibiotic-mediated selection pressure [25]. One of the most well-known bacteria that contributes to the horizontal gene transfer that spreads antibiotic resistance (ABR) determinants and increases the incidence of antibiotic-resistant bacteria is enterococci [26]. This genus is thought to play a major role in the dissemination of ABR genes because it has a range of mobile genetic components, including transposons and conjugative plasmids. Bacteria transfer genes horizontally by three main mechanisms: conjugation, natural transformation, and transduction [27]. Through the horizontal transfer of mobile genetic elements, enterococci have acquired and spread antibiotic resistance. Conjugative plasmids of the pheromone-responsive, broad host range incompatibility group 18 type have mostly mediated this transmission [28].

Conjugation is the process by which one bacteria directly transfers genetic material to another. DNA is passed from one bacterium to another during conjugation. DNA is transferred between cells once the donor cell uses adhesions or cell surface pili to draw itself near to the recipient [29]. The most common mechanism for horizontal gene transfer in nature is thought to be plasmid-mediated conjugation. Since plasmids may reproduce in a variety of hosts and can acquire new genes through mobile genetic elements such as transposons or integrons, they are ideal vectors for the spread of antimicrobial resistance [30]. Since conjugation is inherently aggregative and encourages donor-recipient cell contact, it can be shown that the conjugative plasmid’s core naturally fosters interactions that could eventually result in the production of biofilms [31].

In transformation, a competent bacterium absorbs DNA secreted by other bacteria from its surroundings. A plasmid, which is a circular DNA molecule, is one type of DNA that can be replicated in the receiving cell and passed on to offspring. Biofilms cause higher transformation rates involving both small DNA fragments and big elements, such as plasmids, including those that do not encode genes for mobilization [31]. In transduction, bacteriophages, viruses that infect bacteria, transfer small chromosomal DNA segments “by accident” from one bacterium to another [32]. Accidental transfer of sequence-specific DNA near the chromosomal phage attachment site and subsequent transfer result in specialized transduction.

In comparison to planktonic states, horizontal transfer rates are generally higher in biofilm communities. Additionally, biofilms support plasmid stability and may expand the host range of horizontally transported mobile genetic elements [31]. Plasmids are extrachromosomal genetic components that replicate semi-autonomously.

3. Conclusion

In summary, the genus Enterococcus is notable for its acquired resistance to specific drugs. Due to the ongoing use of antibiotics for medical purposes and as growth promoters in both humans and animals, as well as the existence of insertion sequences, transposons, integrons, and plasmids, these organisms serve as major reservoirs for transferable antibiotic resistance and virulence genes in a variety of ecosystems, including soil, water, and food. For a very long period, both nonpathogenic environmental bacteria and microorganisms that produce antibiotics have had genes for antibiotic resistance.

Through horizontal gene transfer pathways, these resistance genes may be incorporated into clinical bacteria, and since the extensive use of antibiotics in clinical therapy, the rate at which these events occur has rapidly grown. Enterococci appear to have evolved a number of defense mechanisms that increase their likelihood of surviving in the hospital setting. This ability appears to be the outcome of several different elements working together. While the heterogeneity of virulence factors in this genus is unknown, their combination with biofilm formation increases both the susceptibility to adverse conditions and the rate at which infection develops. However, these bacteria pose a threat to public health and antibiotic therapy because of their inherent resistance to a wide range of antibiotics, which is linked to their genome plasticity and ability to acquire new genetic elements related to this resistance. New therapeutic and preventive strategies that aim to stop the spread of enterococci infections may be developed with a deeper comprehension of the mechanisms that allow these pathogens to infect humans as well as the host immune response to them.

Conflict of interest

The authors declare no conflict of interest.