Pathogenicity factors of Acinetobacter baumannii

Introduction

The history of the investigation of the genus Acinetobacter dates back to 1911, when the Dutch microbiologist Beijerinck described a microorganism called Micrococcus calcoaceticus, which was isolated from the soil with a calcium acetate-containing medium [1].

The generic term "acinetobacter" is derived from the Greek words (α (prefix denoting negation), κίνητο (mobility), and βακτηρ (rod)) and is interpreted as "fixed stick". The term reflects the absence of flagellar organelles of movement [1]. The most common species causing infections in humans are A. baumannii, A. calcoaceticus, and A. lwoffii [2]. Results of clinical investigations have shown that A. baumannii is the most pathogenic bacterium of the genus and is one of the leading causes of nosocomial infections throughout the world, including nosocomial pneumonia, especially in people with pre-existing comorbidities [3–5]. A. baumannii is highly resistant to a wide range of antibiotics. The World Health Organization (WHO) classifies this microorganism as a threat to human health worldwide [6].

A. baumannii is a gram-negative aerobic, oxidase-negative microorganism that is often found in soil and water and is also excreted by animals and plants [7][8]. Revealing of its strains may be due to environmental contamination from a primary hospital reservoir or it may originate from natural sources [9].

According to foreign literature, Acinetobacter is one of the six dangerous bacteria included in the ESCAPE group. This term denotes a group of bacteria and is an abbreviation of the first letters of the generic names of bacteria included in this group: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and species of the genus Enterobacter [1].

This microorganism is considered the second most frequently isolated microorganism from clinical material [10]. Risk factors for infection caused by A. baumanii include male gender, advanced age, concomitant diseases of the cardiovascular, respiratory, and other body systems, the duration of the invasive methods of treatment, prolonged stay in a hospital, resuscitation or intensive care units, previous antibiotic therapy with application of cephalosporins, fluoroquinolones, or carbapenems [11]. The clinical significance of A. baumannii, especially over the last 15 years, was stipulated by its distinctive ability to enhance or acquire resistance determinants [12]. Acinetobacter has low virulence, but it can cause infection in immunocompromised patients and neutropenia. Morbidity and mortality in patients with multisystemic organ diseases are high [6].

A. baumannii infections account for about 2% of all infections in the United States and European countries; these indices are twice as high in Asia and the Middle East. Although these infection contamination rates are lower compared to other gram-negative pathogens, nevertheless approximately 45% of all strains are multidrug-resistant throughout the world, and up to 70% in Latin America and the Middle East [13]. In Italy, A. baumannii is responsible for 16.9% of healthcare-associated infections in intensive care units. It was also responsible for 15% of sepsis cases in intensive care units from 2008 to 2017 [14].

Against the backdrop of a pandemic of the new coronavirus infection, the causative agent Acinetobacter spp. is one of the etiological agents that cause the development of community-acquired and nosocomial pneumonia in patients with COVID-19, leading to a more severe disease course [15][16]. Lescure et al. (2020) identified A. baumannii as the causative agent of ventilator-associated pneumonia in a patient infected with SARS-CoV-2. This bacterium is commonly found on medical equipment (including the system used for mechanical ventilation) and is able to survive up to 33 days on dry surfaces [17][18].

Moreover, the acquisition of multidrug resistance by this pathogen, especially to carbapenems, is a major public health problem. Resistance to disinfectants and the ability to produce polysaccharide capsules and biofilms determine the high pathogenetic potential of the bacterium. In 2015 in Greece, 94.5% of strains were resistant to imipenem, while in North American hospitals (2008), 58% of strains were identified as CRAB (carbapenem-resistant Acinetobacter baumannii) [17][18].

The carbapenem-resistant A. baumannii (CRAB) has become world-famous as an important nosocomial pathogen [19]. To date, a significant proportion of A. baumannii strains are carbapenem-resistant (CRAB), that is, they are multidrug-resistant. Carbapenem resistance rates in some countries exceed 90%, wherein mortality from the most common CRAB infections, i.e. nosocomial pneumonia and bloodstream infections, is approaching 60% [20].

The pathogenicity factors of A. baumannii are not only involved in all stages of the infectious process but also ensure the survival of the pathogen, promote tissue injury, and evade the immune system [1].

The high resistance of Acinetobacter spp. strains and their ability to persist and remain active in solutions and on various surfaces create difficulties in choosing adequate antibiotic therapy tactics [21].

Despite the clinical significance of A. baumannii, until recent times there has been limited research on factors contributing to the pathogenesis of this organism. The development of modern molecular biological technologies has allowed specialists to expand their knowledge about the properties of the pathogen. For the first half of 2022 alone, the PubMed system has over 650 citations to publications in leading scientific journals on A. baumannii.

A literature search was carried out in the databases Scopus, Web of Science, RSCI, and MedLine within the period from 1992 to 2022 by key phrases such as "A. Baumannii", "pathogenicity factors", "A. Baumannii outer membrane proteins", "A. Baumannii pili", "A. Baumannii LPS", "A. Baumannii capsule", "A. Baumannii siderophores", "A. Baumannii biofilm production", and "A. Baumannii secretion systems".

The purpose of the literature review was to analyze current literature sources on the pathogenicity factors of A. baumannii and their role in the infectious process. New information on pathogenicity factors will help to better investigate the adaptive potential of A. baumannii under the condition of its impact on the host organism and develop new methods for diagnosing, treating, and preventing diseases caused by this microorganism.

Pathogenicity factors of A. baumannii

Proteins of the outer membrane. Gram-negative bacteria are distinguished by the presence of an additional outer membrane consisting of rather large molecules such as lipopolysaccharides [22]. The membrane also contains proteins. The outer membrane proteins are divided into amphipathic lipoproteins, which provide the connection of the outer membrane with murein, and integral proteins, which perform a structural role. Proteins are constantly synthesized by the cell and make up 80% of the outer membrane proteins [23].

The outer membrane proteins play an important role in the pathogenicity of a microorganism, evading the body's immune response [3] and contributing to the adhesion process in human tissues. There are three types of proteins responsible for attachment to fibronectin: OmpA, TonB-dependent copper receptor, and Omp with a molecular weight of 34 KDa [1].

The outer membrane protein (OmpA) is the main porin protein of the outer membrane of A. baumannii, which is involved in adhesion to host epithelial cells and biofilm production [3]. The OmpA protein can directly induce host cell death when delivered by outer membrane vesicles. After penetration of macroorganisms into the cells, bacteria secrete OmpA, which is able to move into the nucleus and mitochondria and cause releasing of cytochrome C that promotes the translocation of the apoptosis-inducing factor and entails the death of epithelial cells [24][25].

A. baumannii strains isolated from clinical material exhibit low virulence in vivo and do not contribute to a high mortality rate [26].

In addition, OmpA affects the host's immune system. Even though processing A. baumannii with OmpA does not affect the expression level of pro-inflammatory cytokines or chemokines, it does increase the production of nitric oxide synthase (iNOS) and the surface expression of Toll-like receptor 2 (TLR2) in epithelial cells [25].

The A. baumannii OmpA protein stimulates the production of a biofilm on epithelial cells through interaction with fibronectin located on the cell surface [27]. This protein is associated with resistance to carbapenem antibiotics, such as imipenems and meropenems, and remodels autophagy in human epithelial cells [12][28][29].

TonB-dependent copper receptors in gram-negative bacteria are associated with the uptake and transport of large substrates such as iron siderophore complexes and vitamin B12. According to the literature data, removal of the receptors from A. baumannii chromosome results in a reduction of the biofilm produced by a mutant strain with a deficiency of the copper receptor that consequently leads to a decrease in their adhesion to human epithelial cells and hydrophobicity [30].

Pili. Pili are an important adhesion factor, as are proteins associated with the surface membrane. A. baumannii expresses type IV pili required for attachment to host cells. Type IV pili consist of an alone protein subunit called the main pilin, which assembles into a narrow (≈ 6–9 nm) helical fiber of variable length (up to 2.5 μm). Like other Acinetobacter species, A. baumannii does not have flagellas, but exhibits motility dependent on type IV pili. However, the role of these pili has not been fully elucidated. Virstatin, a known inhibitor of type IV pili formation, has been shown to inhibit biofilm formation in A. baumannii. At the same time, another investigation did not demonstrate a correlation between the antigenic variability of the main A. baumannii pilin, pilA, and biofilm formation in vitro [31].

Csu pili, examined in the A. baumannii ATCC 19606 strain, are assembled via the chaperone-asher secretion system. Biofilm production occurs with the participation of Csu pili. The reduction in pili hydrophobicity eliminates bacteria attachment, suggesting that pili tips are used for detection and binding to hydrophobic cavities in substrates. CsuE is located at the pilus tip and is involved in the attachment of bacteria to biotic and abiotic substrates [32].

Lipopolysaccharide (LPS). LPS is the structural component of the outer membrane of Gram-negative bacteria. It consists of a hydrophobic anchor domain called lipid A (or endotoxin), which makes up the external part of the outer membrane of Gram-negative bacteria, an oligosaccharide core, and a specific polysaccharide O-antigen composed of repeating elements. Lipid A is considered the most toxic region of LPS, although the polysaccharide portion of the molecule has potent immunomodulatory and immunostimulatory properties. LPS has been shown to promote bacteria evasion from the host immune system by affecting both innate and acquired host responses to infection and initiating the host's inflammatory response. In addition, the location of LPS on the cell surface facilitates the interaction of the bacterium with the environment [33][34]. The LPSs of A. baumannii strains includes galactose, 2-acetamido-2-deoxyD-galactose, 2-acetamido-2-deoxyD-glucose, 3-deoxy3-(D-3-hydroxybutyramido)-D-quinovose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine [1].

Lipid A is a hydrophobic glycolipid, which is marked by the highly conserved biosynthetic pathway. It is considered important for Gram-negative bacteria. Changes that occur during lipid A biosynthesis through enzyme modifications allow strains to adapt to specific niches. Enzymes provide resistance to certain types of antibiotics and change the permeability of the outer membrane [35].

According to nuclear magnetic resonance spectroscopy data, the polysaccharide is built from repeating trisaccharide units containing α-l-fucosamine, α-d-glucosamine, and α-8- epi-legionaminic acid [36].

Hepta-acylated lipid A is the main species produced by A. baumannii and serves as an anchor for two molecules of 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo or ketodeoxyoctonic acid), which constitute the LPS core region along with the sugar oligomer. The O-antigen can be attached to the core oligosaccharide to form an intact LPS structure. Lipid A and major fragments of Acinetobacter are phosphorylated to varying degrees, generating an overall negative charge of endotoxin molecules called lipolygosaccharides. In addition, the divalent cationic bridges between LPS molecules serve to strengthen the membrane by balancing the electrostatic network [37]

According to recent investigations, mutations in lipid A biosynthesis genes (lpx A, lpx C, lpx D) lead to resistance to polymyxin. The mutation rate in the drug resistance group was 90.45% [38].

Capsule. The carbohydrate structure of the capsule determines the pathogenicity of A. baumannii. The capsule is an evasion factor for innate immunity. For example, genetic injuries in the capsule assembly genes resulting in an acapsular phenotype usually bring on the appearance of a nonpathogenic strain in vivo. In addition, sub-inhibitory concentrations of chloramphenicol increasing the thickness of the A. baumannii capsule raise both pathogenicity and resistance to innate immunity. It is assumed that changes in the structures of the capsule in virulent and avirulent strains affect pathogenicity [37].

The genes required for the biosynthesis and export of exopolysaccharides are grouped at the capsule locus (K-locus). The composition and structure of the capsule vary greatly between A. baumannii isolates [39].

Certain types of capsules have been proven to suppress mammalian defenses in vivo. The capsular polysaccharide is associated with the K-locus and ensures the survival of the microorganism in the human body [40].

It was revealed that the colonies of the A. baumannii 5075 strain could quickly change from opaque (VIR-O) to translucent (AV-T) variant. The VIR-O variant is pathogenic. VIR-O cells have a stronger capsule than AV-T cells and they are more resistant to disinfectants and host immune defense. In addition, 116 genes are differentially expressed between VIR-O and AV-T variants, and any of these genes can affect disinfectant resistance and host immune defense regardless of the capsule [41].

The capsule is a disinfectant resistance factor and provides a survival advantage. However, this mechanism has not yet been identified in vivo. Whether the capsule also serves to protect against attack by phagocytes (eg, neutrophils and macrophages) or antimicrobial peptides remains to be tested [42].

Siderophores. Siderophores are high-affinity iron-chelating molecules synthesized by microorganisms to extract extracellular ferric iron from the environment. The most common siderophore systems revealed in A. Baumannii are baumanoferrin, fimsbactin, and preacinetobactin-acinetobactin (called acinetobactin) [43].

Iron accumulation is important for the A. baumannii pathogenicity and can occur in several ways [3]. Although iron is found in abundance in the environment and biological systems, ferric iron is relatively inaccessible to cells due to poor aerobic solubility and chelation with compounds such as heme and high-affinity iron-binding proteins (lactoferrin and transferrin) [44].

Bacteria have evolved a complex iron uptake system to be able to successfully compete for it under host conditions. The concentration of free iron in bacterial cells is mainly adjusted by the iron uptake regulator Fur. When the concentration of free iron in the cell increases, the Fur protein can bind its ions, thereby inhibiting the genes encoding the uptake system and activating the genes encoding the iron storage protein [45].

A. baumannii does not bind transferrin and does not carry genetic determinants encoding proteins involved in iron uptake from transferrin and lactoferrin.

The strains, in particular ATCC 19606T strain, can use heme as a source of iron by expressing potential iron uptake and utilization systems. The A. baumannii genome contains genes encoding products designed to capture and utilize heme, which can be available to bacteria in regions of severe cell and tissue damage caused by infections, such as necrotizing fasciitis. A. baumannii can also acquire ferrous iron, which is available in low oxygen conditions. A. baumannii contains genes encoding the Feo transport system, the function of which is yet to be examined [44].

Pathogenicity enzymes of A. baumannii. Enzymes can act as invasion factors and catalyze reactions leading to the formation of toxic products and host cell death [1]. Among the virulence factors, the production of extracellular enzymes with lipolytic activity is worth noting. Phospholipases as pathogenicity factors of A. baumannii are the most important hydrolytic enzymes with lipolytic activity against phospholipids of human cell membranes [47]. Enzymes of invasion include phospholipases C and D, proteins with DNase activity and serine protease with anticomplementary activity. Phospholipases contribute to the membrane structure disintegration in the host cells. Proteins with DNase activity are involved in chromosomal DNA injury [1].

The enzyme phospholipase D helps A. baumannii to persist in human serum, which was shown in a mouse model of pneumonia, while another enzyme, phospholipase C, is toxic to epithelial cells [47].

Data on A. baumannii enzymes continue to accumulate. For example, the CpaA enzyme has been identified as a virulence factor that inhibits blood clotting by inactivating clotting factor XII. Thus, CpaA reduces the formation of intravascular clots and promotes the A. baumannii spreading [47].

A. baumannii has enzymes belonging to the carbapenemase group, such as OXA, NDM, VIM, and IMP, which are revealed in clinical strains.

The first OXA enzyme with carbapenemase activity identified in A. baumannii was OXA-23 (first named ARI-1) found in a strain isolated in Scotland. This enzyme gave its name to the first group of OXA enzymes possessing the ability to confer resistance to carbapenems [5].

The presence of the NDM (New Delhi-metallo-beta-lactamases) enzyme stipulates antibiotic resistance to the beta-lactam group, which makes it difficult to treat an infection caused by microorganisms carrying such resistance. It hydrolyzes all beta-lactam antibiotics except aztreonam. The gene encoding NDM-1 is often localized in plasmids and, consequently, is easily transmitted to other microorganisms through horizontal gene transfer, thereby increasing the probability of the emergence of drug-resistant strains of pathogenic microorganisms [48].

The VIM enzyme, or Verona integron-encoded metalloβ-lactamase, has activity against a wide range of β-lactam antibiotics, but cannot hydrolyze aztreonam.

IMP or imipenemase is an enzyme with activity against imipenem metallo-β-lactamase of class B. IMP-positive strains have unique sensitivity profiles, in particular, to ceftazidime and piperacillin-tazobactam. The bla_IMP genes are located in class 1 integrons carried by plasmids and can be spread horizontally among different species [49].

A. baumannii produces 6 types of signaling molecules of N-acylhomoserine lactones. According to the literature, 63% of Acinetobacter produce more than one type of N-acylhomoserine lactones. Synthesis of signaling molecules occurs with the participation of the Aba protein of Acinetobacter from the LuxR family, followed by secretion into the external environment, where they interact with AbaR proteins. The resulting complex N-acyl-homoserine lactone — AbaR binds to the lux-box promoter sequence (in acinetobacteria, lux-box is represented by the CTGTAAATTCTTACAG chain), which regulates the expression of numerous genes and controls the production of pathogenicity factors, locomotor activity, biofilm production, and antibiotic resistance [1].

Biofilm formation. Biofilm formation is an important pathogenic mechanism for many microorganisms including A. baumannii. Numerous factors such as adhesins, capsular polysaccharides, pili, antibiotic resistance, as well as physicochemical parameters (temperature, growth medium, surface hydrophobicity, pH, oxygen concentration), and the presence of other mechanisms, including iron uptake, poly-N-acetyl-β-(1-6)-glucosamine (PNAG)), contribute to the formation and maintenance of A. baumannii biofilms [50].

Due to the ability to produce PNAG, Acinetobacter can form a biofilm at the air-liquid interface when the process is coordinated with the expression of the csuA/B genetic complex that controls pili assembly [51]. The rate of biofilm formation by A. baumannii is 3 times higher than in other Acinetobacter species. In addition, these strains are able to form a biofilm known as a pellicle that increases the surface-bound motility of the bacterium. However, in A. Baumannii, pellicle formation is a rare feature and it is required for the expression of this phenotype. Nevertheless, in the ACB complex (A. baumannii, A. calcoaceticus and the genomic species Acinetobacter 13TU), pellicle formation for A. baumannii was almost four times higher than for other Acinetobacter species.

Biofilm formation in A. baumannii makes it difficult to treat an evolving infection [52]. Despite a large number of works on the association of hospital outbreaks of A. baumannii with severe infections and antibiotic resistance, the factors that determine its virulence and pathogenicity require in-depth additional investigation [50].

Secretion systems of A. baumannii. A. baumannii, similarly to most gram-negative bacteria, express a number of complex secretion systems for the transfer of pathogenicity factors across the cell coat [53][54].

The first secretory system is essential for the autotransport of the surface protein adhesin (Ata). It is revealed in many clinical strains [55]. The type II secretion system (T2SS) is widespread among gram-negative pathogens suitable to live in various conditions, and they use it to export effector proteins [56].

Growth in a medium containing long-chain fatty acids as the sole carbon source requires the protein lipase LipA [57].

T2SS is a two-step process in which proteins with an N-terminal secretion signal move across the inner membrane along the common secretory pathway (Sec) into the periplasmic space. After the removal of the secretion signal, the folded proteins are subsequently secreted into the extracellular space via the T2SS mechanism [56]. Eijkelkamp et al. were the first to report on the availability of T2SS components in A. baumannii [58], which play an important role in colonization when mice are infected. In addition, it remains to be seen whether T2SS effectors cause tissue injuries [56].

Many gram-negative bacteria have a type VI secretion system (T6SS), which is responsible for the ability to transfer protein toxins to other bacteria by contact. T6SS biogenesis and assembly involve the TssA encoding protein; TssB and TssC coat components; Hcp tubule protein; TssE, F, G, and K base plate proteins; TssL and M membrane complex proteins; and ClpV proteins [59].

Conclusion

While A. baumannii is an opportunistic pathogen, the infections it causes are notoriously difficult to treat due to acquired antimicrobial resistance. A. baumannii acquires antibiotic resistance through many different mechanisms.

Adaptation and spreading of the pathogen contribute to its resistance to external environmental factors due to the availability of a capsule and the formation of biofilms, which allows bacterial cells to survive in a hospital environment.

Currently, there are various methods of laboratory diagnostics, both classical (bacteriological method) and the most advanced, which have appeared in recent decades and are widely used in practice, in particular, polymerase chain reaction (PCR).

For PCR diagnostics, a number of test systems are used, for example, AmpliSens® MDR A.b.-OXA-FL, to determine the OXA carbapenemase genes and Acinetobacter baumannii marker genes; RealBest DNA Acinetobacter baumannii/Stenotrophomonas maltophilia (set 1).

A. baumannii has genomic plasticity. Future efforts should be directed toward molecular genetics research. The investigation of the pathogenicity mechanisms of the infectious agent, the emergence of its antibiotic resistance, and evasion of immune defense is the groundwork for the development of new strategies for combating infection caused by A. baumannii.

The application of whole genome sequencing methods is promising for identifying genes representing the markers of A. baumannii strains with increased epidemic potential. It is imperative to investigate the mechanisms of acquisition and transmission of genes encoding factors of pathogenicity and resistance to antibacterial drugs among natural and nosocomial strains.

Genetic testing of clinical strains of A. baumannii, as well as strains isolated from the environment, will provide insight into the molecular mechanisms required for the survival and adaptation of the microorganism and help to identify the most important pathogenicity factors. These factors can serve as potential markers in the development of test systems, which will improve the laboratory diagnosis of diseases caused by A. baumannii.