Key factors of pathogenicity of Enterobacter spp.

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Introduction

Members of the family Enterobacteriaceae, belonging to the order Enterobacterales, are found everywhere: in soil, water, and living organisms. On the one hand, they are commensal microorganisms of the human gastrointestinal tract, and on the other hand, they can cause severe infections of the respiratory and genitourinary tracts, purulent-inflammatory diseases of the skin and soft tissues, and generalized infectious processes. In Russia, between 2010 and 2023, Enterobacterales were the leading cause of both community-acquired and nosocomial infections [1]. These indicators may be associated with the increasing level of resistance of these bacteria to disinfectants and antibacterial drugs, which ensures their widespread distribution in the hospital environment [2]. In 2017, the World Health Organization published a list of microorganisms called ESKAPE, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. In addition to the medical problems associated with severe disease progression, high frequency of complications, and fatal outcomes due to multiple drug resistance, these microorganisms cause significant economic losses. The genus Enterobacter is a group of Gram-negative rod-shaped, facultative anaerobic, non-spore-forming bacteria that are widespread in nature and share many similar morphological, phenotypic, and ecological properties with other members of the family Enterobacteriaceae [3, 4]. The Enterobacter cloacae complex is the group of the greatest clinical significance among Enterobacter spp.

This group consists of 13 heterogeneous genetic clusters according to hsp60 gene sequencing, namely: E. asburiae (cluster I), E. kobei (cluster II), E. hormaechei subsp. hoffmannii (cluster III), E. roggenkampii (cluster IV), E. ludwigii (cluster V), E. hormaechei subsp. oharae and E. hormaechei subsp. xiangfangensis (cluster VI), E. hormaechei subsp. hormaechei (cluster VII), E. hormaechei subsp. steigerwaltii (cluster VIII), E. bugandensis (cluster IX), E. nimipressuralis (cluster X), E. cloacae subsp. cloacae (cluster XI), E. cloacae subsp. dissolvens (cluster XII), and a heterogeneous group of E. cloacae sequences is considered cluster XIII [5]. The most common species of Enterobacter cloacae complex are E. cloacae, E. hormaechei, E. ludwigii, E. asburiae, E. bugandensis, E. kobei, and E. roggenkampii [6].

Highly virulent strains of Enterobacter spp. are often the cause of infections among patients who are hospitalized for long periods (especially in intensive care units), premature infants, and patients with burns, multiple injuries, and diabetes mellitus [7, 8]. The predominant types of biomaterial in which these microorganisms are identified are urine, blood, wound exudate, and sputum [9]. The particular relevance of infections caused by Enterobacter spp. is due to a number of pathogenicity factors that ensure the interaction between the microbe and the host, leading to the development of the disease. The most studied among them are fimbriae, Curli amyloid fibers, pili, and flagella, which provide adhesive activity; type III, V, and VI secretion systems, iron uptake system, which give an advantage in bacterial competition and colonization of ecological niches; toxins, which are clinically important due to their damaging effect on the cells of the macroorganism; capsule formation, providing evasion from immune surveillance; biofilm formation, necessary for bacterial persistence in the hospital environment and resistance to antibacterial drugs.

Pathogenicity factors that enable bacteria to adhere to a macroorganism

Fimbriae

It is known that the development of any infectious process begins with adhesion, which consists of bacteria recognizing receptors on the surface of target cells of a susceptible macroorganism and interacting with them. This is achieved with the help of protein molecules — fimbriae and afimbriae adhesins, which vary in their morphological, functional, antigenic, and biochemical characteristics. Fimbrial adhesins are a component of fimbriae and pili and are most effective in ensuring adhesion. Afimbrial adhesins can be represented by lipopolysaccharide (LPS) in Gram-negative bacteria, outer membrane proteins, or capsular polysaccharides. When studying adhesive ability in experimental in vitro conditions, it was found that E. cloacae strains exhibited the greatest adhesive activity in relation to cells of the urinary tract and lungs. In contrast, adhesion to skin and intestinal epidermal cells was the lowest according to the study. It has been established that type 1 fimbriae are necessary for adequate attachment to target cells during the adhesion process [10]. The main structural unit of type 1 fimbriae is the FimA protein. The terminal adhesin protein FimH not only ensures the adhesion of Enterobacter spp. by binding to mannose-containing receptors on the surface of target cells, but also enables their penetration and the formation of biofilms [11]. Experiments have shown that deletion of the fimA and fimH genes encoding these proteins prevents E. cloacae from adhering to bladder cells, while the absence of CsgA, which is a component of Curli fimbriae, does not affect adhesion [10]. Type 1 fimbriae are present in all members of the family Enterobacteriaceae, including E. coli [12]. It can be assumed that the adhesion process depends not only on the presence of morphological structures for its implementation, but also on the type of microorganism, the nature of the surface, and environmental conditions. In E. hormaechei, type 3 fimbriae play an important role in adhesion, although they are rare among other members of the genus Enterobacter [13]. Type III fimbriae are encoded by the mrkABCDF operon (mrkA, mrkB, mrkC, mrkD, mrkF genes) and are involved in adhesion and biofilm formation on surfaces made of various materials [13, 14]. It should be noted that this pathogenicity factor is present in many members of the family Enterobacteriaceae, since plasmids containing the mrk operon are transferred between Gram-negative bacteria by horizontal transfer [15]. The structural features of type 3 fimbriae are the presence of the major and minor subunits MrkA and MrkF, respectively, with MrkD as the terminal subunit responsible for adhesion [16]. Strains of E. hormaechei subsp. hoffmannii lacking the mrkA gene exhibited lower adhesive activity in vitro not only against Caco-2 colon epithelial cells but also against abiotic plastic surfaces [13].

Curli amyloid fibers

Enterobacter spp., like other members of Enterobacteriaceae, are characterized by the presence of Curli fimbriae, which are extracellular amyloid protein spiral fibers that form connections between themselves, surrounding the cell from the outside [17]. The ability of Enterobacteriaceae to form this pathogenicity factor is associated with the presence of a type VIII secretion system, which uses several proteins to form Curli fibers: CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG, whose synthesis is controlled by the csgBAC and csgDEFG operons. CsgA is the main structural component of Curli amyloid fibrils, and CsgB is an auxiliary subunit [18, 19]. The periplasmic chaperone CsgC ensures the stability of the transitional form of CsgA. The promoter protein CsgD ensures the activation of csgBAC operon transcription [20]. CsgE and CsgF regulate the assembly process of Curli fimbriae [21]. CsgG, moving from the periplasmic space to the outer membrane, forms a pore for the release of the main subunits of Curli fibers [22, 23]. In the presence of the CsgF protein, the nucleator function of CsgB is effectively realized, which ensures the correct polymerization of Curli fibers on the surface of the bacterial cell [24]. Curli fimbriae are an important component of the extracellular matrix in the formation of biofilms, including on the surfaces of medical devices [25]. To persist on the surface of mucous membranes, Enterobacteriaceae have mechanisms that protect them from the harmful effects of the complement system. One of these is the presence of curli fimbriae, which make the bacteria resistant to the classical pathway of complement activation [26]. Furthermore, Enterobacter spp. are characterized by the presence of pili. In a study by F. Brust et al., the papC and papD genes were isolated in E. hormachei, which are responsible for the proteins of the same name involved in the assembly and transport of the main subunits of these bacterial structures [27].

Biofilm formation

Biofilm is a collection of microorganisms that are tightly bound together, attached to a biotic or abiotic surface, and immersed in an extracellular mucous matrix [28]. Their epidemiological danger lies in the fact that they are a reservoir of pathogenic microorganisms, as they can form on the surfaces of medical devices and spread during their use [29]. The ability to form biofilms provides the bacterial community with resistance to antimicrobial drugs and the ability to be inaccessible to components of the human immune system. It is worth noting that biofilm formation is a multi-step process based on several stages [30, 31].

Biofilm formation is characteristic of many Gram-negative bacteria, including Enterobacter spp. It should be noted that biofilm is a dynamic structure that can change depending on environmental factors. A constant increase in the amount of exopolysaccharides in its composition indicates a gradual transition from the adhesion stage to the stabilization of the biofilm, since these components are the basis for the formation of bonds between other chemicals in the microbial community [29]. The wcaA, wcaM, and wza genes identified in E. cloacae are involved in the biosynthesis of biofilm exopolysaccharides [32]. The wcaA gene encodes the enzyme UDP-glucosyltransferase (WcaA), which is necessary for the initiation of the biosynthesis of repeating polysaccharide units. The wcaM gene encodes a glycosyltransferase responsible for the elongation stage of the polymer chain, while the wza gene encodes the Wza lipoprotein, which functions as a channel for polysaccharide export through the outer membrane of the bacterial cell [32, 33]. Nucleic acids are an essential component for stabilizing the biofilm matrix in the early stages [34]. Proteins give biofilms structural stability. In a study by T. Misra et al., it was found that the amino acid tyrosine is produced at a later stage of biofilm formation, indicating their transition to the dispersion phase. The synthesis of valine and proline by biofilm microorganisms is associated with mechanisms of adaptation to metabolic conditions [29]. In a study by S. Liu et al., the ability of Enterobacter spp. to grow in nutrient-deficient conditions due to biofilm formation was revealed [5]. In order to adapt to conditions of nutrient depletion, the amount of lipids in the matrix base increases. Enterobacter spp. isolates from urine were found to form the most stable biofilm [27]. It can be assumed that this is due to the highly developed adhesive ability of Enterobacter spp. to the uroepithelium.

Capsule

The capsule is a surface structure of microbial cells located outside the cell wall or outer membrane and consisting of exopolysaccharides. It performs a number of important functions: it protects against drying out, shields bacterial structures recognized by immunocompetent cells, serves as a source of reserve nutrients, ensures the formation of connections between cells in biofilms, and adhesion of bacteria to target cell receptors. A study of the capsular polysaccharide in E. hormaechei demonstrated its role in protecting against components of the immune system, in particular in preventing the opsonization of blood serum proteins to the surface of microbial cells [35]. A study by X. Qiu et al. showed that different strains of E. hormaechei isolated from clinical material have different capsule variants that affect the pathogenicity of microorganisms [36]. The presence of a capsule in Enterobacter spp. determines its high pathogenic potential, as it increases the survival time of the bacterial population on the surfaces of medical devices by increasing resistance to adverse environmental factors, in particular drying and temperature.

Flagella

Enterobacter spp. are characterized by the presence of flagella, which ensure the motility of bacterial cells. They are thread-like structures, the main component of which is the flagellin protein encoded by the fliC gene. The fliI gene, isolated from representatives of Enterobacter spp., plays an important role in the formation of flagella [37]. The product of the fliI gene is a component of the type III secretion system (T3SS), which is responsible for transporting flagella-associated proteins out of the cell and is an ATPase that releases the energy necessary for T3SS to function. It should be noted that there are five different clusters in the genomes of representatives of the family Enterobacteriaceae, called flagellar loci, flag-1–flag-5. They are responsible for the presence of flagellar systems [38]. Their diversity may explain the differences in the amino acid composition of flagellin in different members of the family Enterobacteriaceae, with alanine, threonine, and serine being the predominant amino acids in the structure of the main flagellar protein [39]. Undoubtedly, bacteria with multiple flagellar systems have an advantage in intermicrobial competition. Flagella are an important factor in the pathogenicity of Enterobacter spp., as they play a key role in the ability to move through biological fluids and tissues, which is necessary for spreading in the host's body and colonizing various organs.

Thus, Enterobacter spp. is characterized by the presence of type 1 and type 3 fimbriae, which are the main structures of the bacterial cell that ensure adhesion to various substrates. Curli amyloid fibers ensure not only the adhesive process, but also the formation of biofilms. Due to the presence of various adhesins, bacteria of the Enterobacter genus form reservoirs of infection on medical devices: urinary and blood catheters, probes, endoscopic equipment, and endotracheal tubes, which increases the risk of nosocomial infections in patients. However, in addition to this, their vital activity requires the presence of pathogenicity factors that ensure colonization of “sterile” loci of the macroorganism for the purpose of developing an infectious process. Their description is presented in the following section.

The main pathogenicity factors of Enterobacter spp. that ensure the adhesion of bacteria in the macroorganism are illustrated in Fig. 1.

 

Fig. 1. Pathogenicity factors that enable bacteria adhesion in a macroorganism.

 

Pathogenicity factors ensuring colonization activity and survival in ecological niches

Type V secretion system

The type V secretion system (T5SS), or auto-transporter system, delivers effector proteins across the outer membrane without the need for complex multi-subunit complexes [40]. A distinctive feature of T5SS is the formation of a cylindrical structure in the periplasmic space from part of the secreted polypeptide, which acts as a pore through which the protein exits. T5SS auto-transporters perform a variety of functions: adhesion to target cells, proteolytic activity of IgA proteases, and biofilm formation. The literature describes the isolation of the pic gene in E. cloacae, which encodes a high-molecular-weight auto-transporter protein (protein involved in colonization, Pic) related to T5SS [32]. The Pic protein, possessing serine protease activity, acts on high-molecular-weight glycoprotein mucins (MUC2, MUC5AC), which form a dense gel-like layer on the surface of the intestinal mucosa and urinary tract. The destruction of the mucin structure facilitates bacterial adhesion and penetration into deeper tissues, and the products of mucin degradation can serve as a source of carbon and nitrogen for bacteria, providing them with nutrients in competition with the microbiota of these loci. Despite its mucolytic activity, the Pic protein stimulates mucin secretion by goblet cells in the intestine [41]. Furthermore, Pic can inactivate the C3/C3b component of the complement system, suppressing opsonization and the formation of the membrane attack complex, which allows E. cloacae to evade the mechanisms of innate immunity.

Type VI secretion system (T6SS)

The type VI secretion system (T6SS), which is a complex of proteins in the membrane structure of Gram-negative bacteria, is necessary for bacteria to ensure their survival through microbial competition for colonization of ecological niches and food resources [42]. With its help, the microbial cell synthesizes effectors — enzymes (in particular, nucleases) and pore-forming toxins — in response to the appearance of a competing microorganism nearby. Representatives of family Enterobacteriaceae have proteins that differ in their mechanism of action. For example, E. coli has catalase (KatN), which contains manganese ions and reduces the concentration of active oxygen forms inside target cells, which contributes to the intracellular survival of bacteria, while VgrG1 participates in bacterial adhesion and evasion of the host's innate immune system [43, 44]. T6SS provides the formation of a channel in the cell membrane structure through which synthesized substrates enter the target cell. Representatives of the genus Enterobacter also have this secretion system [45]. The key genes of the T6SS system are clpB, icmf, and VasD/Lip, which encode the structural and regulatory components of the system [32]. The clpB gene encodes a bacterial molecular chaperone that has the ability to disaggregate stress-denatured proteins together with the DnaK system, protecting bacteria from high temperatures, acidity, and oxidation [46]. The VasD/Lip gene encodes the Hcp (hemolysin coregulated protein) protein, which forms hexameric rings that assemble into a hollow tubular rod (needle) of the T6SS. The literature describes the possibility of the presence of various gene clusters in Enterobacter spp. — T6SS-A, T6SS-B, and E. cancerogenus has been found to have the T6SS-C type. One strain of Enterobacter spp. can simultaneously have several variants of the type VI secretion system encoded by the presented gene clusters, which is due to their horizontal transfer between different representatives of family Enterobacteriaceae [47]. In a study by J. Soria-Bustos et al., T6SS-1 provided hemolytic activity in E. cloacae, while T6SS-2 participated in adhesion to surfaces of various natures and biofilm formation. It was also found that neither T6SS-1 nor T6SS-2 in E. cloacae provided anti-phagocytic activity [48].

Iron absorption system

Iron is essential for bacterial life due to its involvement in many metabolic processes. It is a component of catalase and superoxide dismutase, which provide protection against oxidative stress. For this reason, bacterial cells have learned to absorb trivalent iron (Fe(III)) complexes from the host organism by producing chelating agents called siderophores [49]. Several classes of siderophores are distinguished depending on the chemical structure of the iron-binding fragments: catecholate, hydroxamate, carboxylate, phenolate, and mixed types. It should be noted that the transport mechanisms of the siderophore complex bound to Fe(III) (Sid-Fe³⁺) in Gram-positive and Gram-negative bacteria differ due to differences in cell wall structure. The movement of Sid-Fe³⁺ into the cell in Gram-negative bacteria is called ROSET (Rotational Surveillance and Energy Transfer), since the resulting complex is unable to penetrate the bacterial cell by simple diffusion or through membrane pores due to its large size [50]. Enterobacteriaceae recognize the Sid-Fe³⁺ complex thanks to specific outer membrane receptors (OMR). Despite differences in OMR among different bacteria, they all interact with the inner membrane protein TonB, whose physical rotation, with the participation of the inner membrane proteins ExbB, ExbD leads to conformational changes in the FhuA (ferric hydroxamate uptake protein component A) protein, which is the main outer membrane receptor for siderophore binding in Gram-negative bacteria [51]. As a result, Sid-Fe³⁺ is released into the periplasm, binds to the ABC transport complex of the inner membrane, and is transported into the cytoplasm, where iron ions are released.

The innate immune response in humans has mechanisms to protect against iron uptake by bacterial siderophores. One of them is the production of lipocalin-2 (a protein associated with neutrophil gelatinase) during the acute phase of the infectious process. It has a bacteriostatic effect because it binds catecholate-type siderophores, which makes it impossible for the host organism to use iron due to the cessation of microbial growth [52]. Under experimental conditions, it has been proven that lipocalin-2 deficiency leads to increased bacterial division and more severe respiratory infections [53]. Enterobacter spp. is characterized by the presence of clusters of genes entABES, iutA/iucABCD, iroBCDEN, encoding enterobactin, aerobactin, and salmochelin, respectively [36]. The entA, entB, and entE genes are responsible for the synthesis of enterobactin from chorizmic acid, a precursor formed in the shikimate pathway. The product of the entS gene is responsible for the export of synthesized enterobactin from the cell. The iron-enterobactin complex is recognized and transported into the bacterial cell by specific transporters such as FepA, FepB, FepC, FepD, and FepG [54]. The iutA protein is an integral β-barrel protein of the outer membrane of Gram-negative bacteria. It acts as a highly specific receptor for the aerobactin-iron (Fe³⁺) complex [12, 15, 37]. The ybt gene cluster encoding yersiniabactin was initially identified in the High Pathogenicity Island (HPI) of Y. enterocolitica [15, 55]. The irp1 and irp2 genes, ybtS, isolated from E. cloacae, are responsible for the biosynthesis of yersiniabactin [15, 55]. Y. enterocolitica [15, 55]. The irp1 and irp2, ybtS genes isolated from E. cloacae are responsible for the biosynthesis of yersiniabactin, while fyuA is both a receptor for the siderophore yersiniabactin and the bacteriocin pesticin [5]. The protein encoded by the fyuA gene belongs to the group of TonB-dependent receptors [32]. FyuA biosynthesis is controlled by the Fur protein, a global iron regulator, and is activated when iron concentrations decrease. The irp1, irp-2, and fyuA genes are often located on mobile genetic elements, particularly HPI, which allows them to be easily transferred between different bacteria, spreading highly virulent properties [12, 14, 56]. Salmochelin is a glycosylated form of enterobactin, and the iroB gene plays an important role in this process. After the iron-salmochelin complex returns to the bacterial cells, iron ions are released and salmochelin is cleaved by esterase encoded by iroD. The iroBCDEN gene cluster is mainly localized on virulence plasmids, which is important for the spread of virulence genes among Enterobacteriaceae. It can be concluded that the ability to synthesize siderophores, inherent in Enterobacter spp., provides the possibility of bacterial competition for food resources and ecological niches.

Siderophores play a key role in the formation of the pathogenic potential of enterobacteria, providing a competitive advantage in conditions of iron deficiency in the macroorganism. The introduction of the Kleborate bioinformatic analysis system has made it possible to standardize the identification of hypervirulent strains of Klebsiella pneumoniae based on the detection of key genetic determinants of virulence. The criterion for hypervirulence in this system is the presence of genes encoding high-affinity siderophores: aerobactin (iuc), salmochelin (iro), yersiniabactin (ybt), as well as the clb operon responsible for the synthesis of the genotoxin colibactin, which also has chelating activity. A comprehensive analysis of these markers provides an integral assessment of the pathogenicity of the strain, determining its belonging to the hypervirulent phenotype [57]. Given that all of the genetic determinants listed are conservative and widespread among members of the family Enterobacteriaceae, including Enterobacter spp., the Kleborate methodology appears to be highly promising for assessing virulence in this group of microorganisms. Similar technologies for molecular genetic detection and assessment of the most aggressive strains expand the possibilities of personalized medicine. It is known that a number of pathogenicity factors (e.g., colibactin in members of the family Enterobacteriaceae) are important in the formation of the oncogenic potential of microorganisms. Consequently, molecular genetic detection of such factors can be used in non-invasive diagnosis of cancer [58].

A key factor in the survival of Enterobacter spp. in the host organism is its ability to resist oxidative stress induced by neutrophils and macrophages. The main bactericidal agents in this process are reactive oxygen species, namely superoxide anion. The first line of defense against this powerful oxidant is the enzyme superoxide dismutase. In Enterobacter spp., as in many other bacteria, the main cytoplasmic isoform of this enzyme is iron-dependent superoxide dismutase, SodB [32]. Expression of the sodB gene is activated by iron deficiency under the action of the global regulator Fur. The rpoS gene, isolated from E. cloacae, encodes an alternative σ-factor that regulates the response of bacteria to stress and the expression of certain genes associated with the virulence of microorganisms [37, 59]. The expression of the rpoS gene is enhanced under unfavorable environmental conditions. Thus, the rpoS gene activates the synthesis of catalases (KatE, KatG) and peroxidases, providing protection against hydrogen peroxide and other active forms of oxygen produced by phagocytes; regulates the synthesis of osmoprotectants; and promotes the formation of biofilms [60]. Thus, the rpoS gene is responsible for the ability to reprogram cellular metabolism into “survival mode,” which directly affects pathogenic potential, biofilm formation, and the effectiveness of antimicrobial therapy.

Thus, the high pathogenicity and successful colonization of ecological niches by representatives of the genus Enterobacter are ensured by a complex and multi-level arsenal of pathogenicity factors. Bacterial secretion systems of types V and VI are necessary not only to evade immune system factors, but also to compete with representatives of the normal human microbiota for colonization of loci and food resources. The presence of multiple siderophore systems (enterobactin, aerobactin, salmochelin, yersiniabactin), encoded by both chromosomal and plasmid genes, gives Enterobacter spp. a decisive advantage in competition for iron ions with other microorganisms and phagocytes. The enzymatic antioxidant defense system, which includes catalases, peroxidases, and superoxide dismutases, reduces the susceptibility of bacterial cells to oxidative stress.

The main factors of Enterobacter spp. pathogenicity, ensuring colonization activity and survival in ecological niches, are illustrated in Fig. 2.

 

Fig. 2. Pathogenicity factors ensuring colonization activity and survival in ecological niches.

 

Structural components of bacterial cells involved in pathogenicity

Outer membrane proteins

Although not classic virulence factors, outer membrane proteins make a decisive contribution to bacterial survival by mediating evasion of the immune response, transport of molecules (including antibiotics), and the formation of antimicrobial resistance. The outer membrane of Gram-negative bacteria has a complex structure, including phospholipids in the inner layer and LPS in the outer layer. The outer membrane is permeated by hydrophilic pores through which water and hydrophilic molecules pass by passive diffusion. The proteins that form these channels are called porins, or outer membrane proteins (OMP). There are various structurally different non-specific OMPs: OmpA, OmpC, OmpF, OmpX, which are constantly expressed in bacterial cells. Specific OMPs include the proteins LamB, PhoE, FhuA, and FepA. Their synthesis occurs in the presence of a specific substrate or under the influence of environmental factors, such as the presence of antibiotic molecules. Modifications of OMPs can lead to the formation of multiple drug resistance in bacteria [3].

OmpA is the main β-cylindrical structural protein that forms a connection between the outer membrane and the underlying layer of peptidoglycan [61]. In addition, OmpA is important for protecting bacterial cells from the effects of antibiotics and toxins, regulating osmotic balance through the transport of Na⁺, K⁺, H⁺, and nutrient absorption. According to the literature, OmpA is a receptor for bacteriocins and bacteriophages. OmpA is capable of interacting with proteins on the surface of neutrophils to suppress the expression of Toll-like receptor 4 (TLR4) and enhance the expression of TLR2. In this way, bacteria penetrate macrophage cells and avoid destruction by inhibiting the synthesis of active forms of oxygen. In the vast majority of representatives of the family Enterobacteriaceae, the role of OmpA and OmpX proteins in the formation of biofilms on abiotic surfaces has been established [62, 63]. In a study by H. Hirakawa et al., the role of the OmpX protein in the formation of flagella in E. coli was established, since the deletion of the ompX gene led to a decrease in the motility of the microorganism due to a reduction in the number of flagella [64]. In a study by M. Mishra et al., Enterobacter spp. isolates isolated from the environment and possessing OmpA and OmpX proteins exhibited multiple drug resistance compared to isolates that did not possess them. It is interesting to note that in clinical isolates of Enterobacter spp., the relationship between the presence of OMP and antibiotic resistance is less common due to a variety of other mechanisms of drug resistance formation [3].

The function of the OmpC protein is not only to regulate the permeability of the outer membrane, adapting to stressful conditions, but also to transport antibiotic molecules into the bacterial cell [65].

The integral protein LamB, which belongs to the porin family, facilitates the diffusion of maltose and maltodextrins through the outer membrane of bacterial cells of the family Enterobacteriaceae. In addition, the transmembrane protein may be involved in the transport of glucose, lactose, and glycerol. It is specific because its expression occurs under conditions of maltose deficiency in the environment. It has been studied that in E. coli, LamB is a receptor for bacteriophage ƛ [66].

The outer membrane proteins of Enterobacter bacteria are key structural components located at the interface between the pathogen and the environment and host organism. They form porins for the passive diffusion of small molecular weight molecules and participate in adhesion and biofilm formation. Changes in the expression of outer membrane proteins are a mechanism for regulating the cellular transport of food resources, toxins, and antimicrobial molecules. In addition, outer membrane proteins are pathogen-associated molecular patterns (PAMPs) recognized by Toll-like receptors, which initiate the development of immune responses in the host organism.

The main structural components of the Enterobacter spp. bacterial cell involved in the realization of pathogenicity are illustrated in Fig. 3.

 

Fig. 3. Structural components of bacterial cells involved in pathogenicity.

 

Toxins

The most important factor in the pathogenicity of Enterobacter species is the secretion of a wide range of exotoxins—protein molecules that cause pathological changes in the macroorganism. These compounds not only suppress the host's immune response, ensuring the survival and reproduction of bacteria, but also directly damage tissues and organs by disrupting cellular processes. Furthermore, the production of bacteriocins, which are a type of toxin, is one of the mechanisms of competition between different species of microorganisms for resources.

Some representatives of Enterobacter spp. produce cytotoxic necrotizing factor type 1 (CNF1) [32, 67]. This toxin causes the activation of small Pho family GTPases, which affect various metabolic cellular processes [68]. They play an important role in cell proliferation, cell apoptosis, and regulation of the actin cytoskeleton. CNF1 contributes to the reorganization of the cell cytoskeleton, which is responsible for maintaining the shape of microbial cells, organizing membrane structures, synthesizing murein as a component of the cell wall, and mobility. CNF1 promotes the production of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α, in cells of various origins, such as T lymphocytes, dendritic cells, and monocytes. In addition, the toxin enhances the transcription of cyclooxygenase-2, as well as the cell adhesion molecule ICAM-1. Increased cyclooxygenase-2 activity leads to the synthesis and accumulation of prostaglandins, which are responsible for the development of inflammation, edema, and pain. CNF1 production increases the risk of infection because it enhances the ability of bacteria to colonize the epithelium and reduces the phagocytic activity of macrophages [69, 70].

Enterobacter spp. produce a secreted autotransport toxin encoded by the sat gene [67]. This pathogenicity factor has a cytopathic effect, which is particularly pronounced in endothelial cells and the urinary tract. This fact suggests that the toxin may be involved in various stages of the pathogenesis of bacteremia and sepsis. In vitro laboratory studies have shown that the serine protease Sat has high proteolytic activity against complement system components and can inhibit their activation in various ways [71]. The involvement of the Sat toxin in the destruction of tight junctions in the epithelial cells of the intestine and kidneys contributes to the infection of these loci by Enterobacter spp. bacteria.

Bacteria of the genus Enterobacter possess hemolytic activity. Under experimental conditions, E. cloacae produced enterotoxic hemolysin [52]. The main role of hemolysin is to lyse red blood cells, which leads to the release of iron-containing heme molecules that are absorbed by bacterial cells to maintain their vital functions. Hemolysin increases the invasion of pathogenic microorganisms into tissues. It is important to note the stimulating effect of hemolysin on immunocompetent cells that produce pro-inflammatory cytokines. E. cloacae can produce cytotoxic enterotoxins [53]. Under experimental conditions in laboratory animals, it was found that these toxins secreted by microorganisms can cause lysis of phagocytes and epithelial cells. The enterotoxic activity of Enterobacter spp. consists in the ability to induce apoptosis of the intestinal epithelium [72]. E. cloacae is capable of producing bacteriocins [73, 74]. These are necessary to suppress the growth of other bacteria in the environment and provide a competitive advantage for their hosts. Bacteriocins inhibit the growth of potential competitors, which is especially important in conditions with a high density of microbial environment and a limited amount of nutrients.

LPS, which is an endotoxin, is an important structural component of the outer membrane of Gram-negative bacteria, providing a barrier function. LPS consists of a hydrophilic O-antigen (or O-polysaccharide), a major oligosaccharide (also hydrophilic), and lipid A. LPS biosynthesis is a complex, multi-step process controlled by enzymes encoded by genes in the waa locus. In E. cloacae, the waaL and waaG genes, which are responsible for the final stage of complete LPS molecule formation, have been identified [32]. The waaG gene is responsible for the formation of the oligosaccharide core by encoding a glucosyltransferase that catalyzes the transfer of a glucose molecule from an activated donor form (UDP-glucose) to the growing chain of the LPS oligosaccharide core [75]. The waaL gene encodes the O-antigen ligase enzyme, which catalyzes the final stage of complete LPS biosynthesis — the covalent attachment of the O-antigen polysaccharide to the oligosaccharide core [76]. It should be noted that, despite the uniform basis of the LPS structure, there are modifications of its components in different species and strains of Gram-negative bacteria [77]. Such changes in LPS can affect its immunogenicity and ability to evade recognition by the host's immune system. Endotoxin is released when bacterial cells die. It is one of the key factors in the pathogenicity of Enterobacter spp. for several reasons. First, LPS serves as a powerful immune stimulant, activating macrophages and dendritic cells through binding to Toll-like receptors (TLR4). This interaction initiates a signaling cascade leading to the activation of nuclear factor kappa B (NF-κB) and the subsequent production of proinflammatory cytokines—TNF-α, IFN-γ, IL-1β, IL-6 [78]. Endotoxin is an exogenous pyrogen. LPS can induce a shock response through mediators involved in the development of a systemic inflammatory response and subsequently sepsis.

Thus, the ability of Enterobacter spp. to produce toxins is crucial in the development of the clinical picture of the infectious process — from local damage to systemic, life-threatening conditions such as septic shock. Endotoxin, which is the LPS of the outer membrane of Gram-negative bacteria, participates in the progression of the systemic inflammatory response by stimulating the massive release of pro-inflammatory cytokines, which in turn leads to vasodilation, increased vascular permeability, hypotension, disseminated intravascular coagulation, and multiple organ failure. The exotoxin CNF1 facilitates invasion and resistance to phagocytosis by members of the genus Enterobacter due to the reorganization of the cytoskeleton of eukaryotic cells. The hemolytic activity of Enterobacter spp. increases the concentration of free iron in the environment, which can be used for bacterial growth with the help of siderophores. Bacterial toxins are powerful determinants of pathogenicity, which not only cause damage to the tissues of the host organism, but also actively modulate the host's immune response, often determining the outcome of the infectious process.

Enterobacter spp. toxins are illustrated in Fig. 4.

 

Fig. 4. Enterobacter spp. toxins.

 

Conclusion

It is difficult to overestimate the clinical significance of representatives of the family Enterobacteriaceae, which are among the main causative agents of healthcare-associated infections in recent years, both in Russia and worldwide.

The analysis of the literature data demonstrates that the high pathogenic potential of representatives of the family Enterobacteriaceae, in particular the genus Enterobacter, is due to a wide range of pathogenicity factors (table) that sequentially ensure adhesion, colonization, evasion of the immune response, and the development of the infectious process. Thus, type 1 and 3 fimbriae, Curli amyloid fibers, pili, and flagella determine the initial stage of interaction with host cells—adhesion. Secretion systems of types III, V, and VI, acting on the cells of the macroorganism, indirectly create advantages for Enterobacter spp. colonization. At the same time, the production of siderophores allows them to effectively compete with the autochthonous microbiota for vital resources such as iron. The ability to form biofilms determines the long-term persistence and successful colonization of bacteria in the hospital environment, forming a stable reservoir of nosocomial infections. Bacterial toxins contribute directly to the development of the clinical picture and severe complications. Thus, the success of Enterobacter species as pathogens is ensured by the combined action of many factors involved in the various stages of the infectious process.

 

Genetic determinants of pathogenicity factors in Enterobacter spp.

Pathogenicity factor category	Pathogenicity factor	Gene	Pathogenicity role	Sources
Adhesins and invasins	Type 1 fimbriae	Operon fim	Adhesion, colonization, invasion	[10, 11]
	Type 3 fimbriae	Operon mrk	Adhesion, biofilm formation	[13]
	Curli fimbriae	Operons csgBAC and csgDEFG	Adhesion, formation of extracellular matrix biofilms	[18-24]
	Pili	papC, papD	Adhesion	[27]
Secretion systems	T3SS	fliI	Release of energy for transporting flagellum-associated proteins out of the cell	[37]
	T5SS	pic	Adhesion to target cells, proteolytic activity of IgA proteases, and biofilm formation	[32, 41]
	T6SS	clpB, icmf, VasD/Lip	Synthesis of effectors for interbacterial competition, action on eukaryotic cells; extraction of metal ions from the environment for persistence	[32, 42]
Iron absorption systems	Enterobacterin	Operon entABES	Acquisition of iron ions from compounds with transport proteins, protection from active forms of oxygen	[5, 36]
	Aerobactin	Operon iucABCD-iutA
	Salmochelin	Operon iroBCDEN
	Yersinia bacterin	irp1, irp2, ybtS, fyuA
Transmembrane transport	Outer membrane proteins	ompA, ompX, ompC, lamB	Maintaining the structure of the bacterial cell; transporting various molecules across the outer membrane into the bacterial cell; forming biofilms; receptor function	[61-66]
Toxins	Hemolysin	hlyC, hlyD	Hemolytic activity	[52]
	Colicin	Operons of endonuclease clb	Interbacterial competition	[73, 74]
	Endotoxin (lipopolysaccharide)	waaL, waaG	Structural integrity of bacterial cells; barrier function; induces immune response; development of shock	[32, 75, 76]
	Type 1 cytotoxic necrotizing factor	cnf1	Activation of GTP-binding proteins of the Rho family; release of proinflammatory cytokines	[32, 67-70]
	Secreted autotransport toxin	sat	Cytopathic effect expressed in relation to endothelial cells and urinary tract cells	[67, 71]
Mobility	Flagella (flagellin protein)	fliC	Mobility, adhesion	[39]
Protection against oxidative stress	Superoxide dismutase	sodB	Protection of bacterial cells from reactive oxygen species	[32]
	σ-factor RNA polymerase	rpoS	Activates the synthesis of catalases and peroxidases; regulates the synthesis of osmoprotectants; promotes the formation of biofilms	[37, 59]

 

This review systematizes data on the genetic determinants of pathogenicity in members of the family Enterobacteriaceae, particularly Enterobacter spp., as well as their clinical and epidemiological significance. A deep understanding of the molecular mechanisms of pathogenicity serves as the basis for the development of fundamentally new approaches in clinical practice. The creation of diagnostic test systems based on polymerase chain reaction and high-throughput sequencing allows us to move towards personalized rational antimicrobial chemotherapy. It also becomes possible to assess the fundamental need for anti-epidemic and clinical measures. The introduction of these technologies is aimed at solving key health care problems: preventing unjustified empirical antibiotic therapy, reducing the burden of antibiotic resistance, and helping clinicians prevent infections from becoming chronic, aggravating their course, and developing complications.

About the authors

Daniil A. Kokorev

Samara State Medical University

Author for correspondence.
Email: d.a.kokorev@samsmu.ru
ORCID iD: 0000-0002-9991-6750

junior researcher, Laboratory of genetic technologies in microbiology, Professional Center for Education and Research in Genetic and Laboratory Technologies, assistant professor, Chair of medical microbiology and immunology

Russian Federation, Samara

Ekaterina A. Strazhina

Samara State Medical University

Email: e.a.strazhina@samsmu.ru
ORCID iD: 0009-0000-1954-9245

specialist, Laboratory of educational technologies in genetics, microbiology and laboratory diagnostics, Professional Center for Education and Research in Genetic and Laboratory Technologies

Russian Federation, Samara

Artem V. Lyamin

Samara State Medical University

Email: a.v.lyamin@samsmu.ru
ORCID iD: 0000-0002-5905-1895

Dr. Sci. (Med.), Associate Professor, Director, Professional Center for Education and Research in Genetic and Laboratory Technologies, Professor, Chair of medical microbiology and immunology

Russian Federation, Samara

References

Supplementary files