Acinetobacter baumannii Virulence Factors, Resistance Mechanisms, and New Insights on Infection Treatment

Acinetobacter baumannii is an alarming pathogen that threatens human health around the world and most of the antibiotics have become unable to cope with it. It has been classified by the World Health Organization (WHO) as a member of the most dangerous ESKAPE organisms “ Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae ” which show high resistance rates toward antibacterial agents. A. baumannii causes life-threatening infections with limited treatment options that include pneumonia, bacteremia, meningitis, urinary tract infection, and wound infection, and numerous reports have documented A. baumannii infection after SARS-CoV-2 infections in multiple publications through the COVID-19 catastrophe. Many virulence factors such as efflux pumps, outer membrane proteins (OMPs), phospholipase, lipopolysaccharide, capsule, protein secretion systems, nutrient-acquisition systems, biofilm production, and quorum sensing account for the pathological and lethal effects of A. baumannii . The present review concentrates on highlighting the major mechanisms of antibiotic resistance declared in A. baumannii , the virulence factors of A. baumannii, and the novel therapeutic strategies. These strategies include novel antibiotics, drug repurposing, antimicrobial peptides (AMPs), nanoparticles, bacteriophage therapy, monoclonal antibodies of humans (Hu-mAbs), and gene amendment in an attempt to help the scientific research society.


Introduction
Antimicrobial resistance of A. baumannii has arisen as a triggering and substantial disaster that increases health system expenditures throughout the world. In current years it has been linked to abundant mortality, infections, and increased expenditures because of both the long duration of treatment and hospitalization. During the previous decades several multicenter studies data have declared that both outpatient-acquired and hospital-acquired antimicrobial resistance are upgrading with the elevated number of immunodeficient older patients [1, 2].
A. baumannii was originally described as Micrococcus calco-aceticus and was first isolated in 1911 by the dutch microbiologist Beijerinck from soil using media enriched with calcium acetate [3]. To differentiate it from the genus Achromobacter motile organisms the genus Acinetobacter comes from the "akinetos", a Greek word [4]. By 1968 the genus Acinetobacter was accepted after Baumann et al., a comprehensive study of organisms such as Moraxella lwoffi, Micrococcus calco-aceticus, Mima polymorpha, Alcaligenes hemolysans, Bacterium nitrate, and Herellea vaginicola, which could not be further sub-classified into variable species and were confirmed to belong to a single genus depending on the phenotypical characteristics [5].
The genus Acinetobacter comprises strictly aerobic, Gram-negative, non-fermenting, nonmotile, non-fastidious, oxidase-negative, and catalase-positive bacteria with a 39% to 47% percent DNA G + C content [6]. The use of DNA-DNA hybridization technique had a great role in the identity establishment of almost thirtythree various Acinetobacter genospecies [7,8]. Among these genospecies, the ACB complex (A. calco-aceticus, A. baumannii, and Acinetobacter genomic species 13TU), show phenotypic characteristics that make them difficult to be distinguished and they represent the most clinically important members of the genus [9]. A. baumannii compromises the highest clinical importance of the ACB complex, while A. calcoaceticus is seldomly described as a cause of huge pathological disorders and has been considered an environmental pathogen [10].
One of the most important nosocomial pathogens is A. baumannii which was classified by the WHO organization as a member of the most dangerous ESKAPE organisms [11]. Numerous reports have documented A. baumannii infection after SARS-CoV-2 infections in multiple publications through the COVID-19 catastrophe [12]. During the 1970s, A. baumannii clinical isolates were sensitive to commonly used antimicrobials, such as gentamicin, ampicillin, nalidixic acid, and chloramphenicol, while, by the end of 1970s, it arises as a serious pathological agent of hospital-acquired infections, especially with the use of broad-spectrum antimicrobials in medical care units [8]. Nowadays, it acquires a high nonsusceptibility rate toward the majority of first-option antimicrobials, however, tigecycline & colistin has become the reserved antibiotics for multidrug-resistant (MDR) A. baumannii treatment; unfortunately, colistin-resistance has been detected in various areas worldwide [13]. The carbapenem resistance rate of A. baumannii has increased by two-fold approximately beside its resistance to various antibiotic classes such as macrolides, fluoroquinolones, and cephalosporins, which has been declared to reach 80% in certain countries and 56% in others from 2005 till 2018 which is considered a great disaster [14].
Li-Kuang et al. revealed that the nonsusceptibility rates of A. baumannii clinical isolates to quinolones (levofloxacin & ciprofloxacin) and sulfonamides were 73.6% and 71.3%, respectively. It showed more than half (50-70%) resistance rates to beta-lactam/betalactamase inhibitor combinations (piperacillintazobactam), cephalosporins (cefepime and ceftazidime) and carbapenems (imipenem, meropenem, and doripenem). However, it showed a 26.7% nonsusceptibility rate toward the tigecycline and 0% of the isolates were nonsusceptible to colistin [15]. A. baumannii is considered a major cause of about 2% of nosocomial disorders in Europe and the United States in 2011 [16]. About 45% of A. baumannii are considered MDR microorganisms, which was 4x higher in comparison to other Gm-ve microorganisms, like Klebsiella pneumoniae and Pseudomonas aeruginosa. The MDR incidence rate of A. baumannii compromised about 70% in the Middle East and Latin America [17]. The WHO has considered A. baumannii which showed carbapenem-resistant as a critical group of pathogens that implement a crucial threatening to humanity's biosafety, triggering the significant demand for novel antimicrobial agents [18].

A. baumannii pathological manifestations
The likelihood of patients being colonized and medical equipment being contaminated by A. baumannii is mainly because it is ubiquitous and is carried on the skin of health care workers and can survive on dry surfaces for up to a month [19]. There are multiple Acinetobacter species; all can lead to human diseases, but mainly A. baumannii accounts for nearly 80% of infections [20]. The majority of A. baumannii disorders target mainly human body organs that are rich in fluid content, like the respiratory system, abdomen peritoneal cavity, and urinary tract. The recurrence of A. baumannii infections in any hospital is considered an alarm of dangerous illness, with about a thirty percent mortality rate [21]. Some of these infections are listed below.

Pneumonia
The adherence and biofilm formation of A. baumannii to the hospital devices such as the endotracheal tube and the creation of a niche for the rapid dissemination of the microbial cells resulted in the increase of carbapenem-resistant Acinetobacter incidences in the intensive care units (

Virulence factors of Acinetobacter and pathogenesis
A. baumannii has many virulence factors involved in its infection (Fig. 1) including:

Motility
A. baumannii has two types of motility, surface-associated and twitching motility that help it to survive and spread on surfaces although being famous as a non-motile micro-organism Studies showed that this crucial pathogen had these two types of motility resulting in an increase in its virulence [33, 34].

Lipopolysaccharides
A. baumannii lipopolysaccharides (LOS) that include only, lipid A and core oligosaccharide, are strong enhancers of circulating WBCs to produce proinflammatory reagents, which are cytotoxic to neutrophils and suppress their migration and their phagocytic ability [54]. The lipopolysaccharide virulence factor has become a crucial factor in the survival of the microorganism because of its vital role in the induction of proinflammatory mediators and colonization. It was declared that modification of lipopolysaccharides can result in declining antibiotic susceptibility [55]. Capsular polysaccharides that are located around the surface of the bacteria act as a defense hail against environmental strains and some antibiotics, so they have a definitive participation in the stays of the pathogen mainly in serum, which has a crucial role in preventing phagocytosis of the microorganism [54, 56].

Biofilm formation
A. baumannii can use biofilm-associated proteins (Bap) in the development of biofilm structure as a result of stressful conditions. Biofilm formation assists bacterial cell colonization and its survival through the adherence to both living and nonliving surfaces leading to hospital device-linked diseases in medical facilities. It was documented that QS (quorum sensing) is concerned with biofilm production by auto-inducers such as signaling molecules [57]. As well as Csu pili and type IV pili are crucial for biofilm formation [58]. It was concluded that biofilm enhances the survival time of A. baumannii in dry areas [59]. Desiccation withstanding refers to the capability to be viable during dry circumstances and it is variable between the A. baumannii clinical isolates, as it can be up to a hundred days withstanding. This ability isn't yet fully defined and is multifactorial [60, 61].

Phospholipases
Phospholipase C and phospholipase D are considered as another crucial virulence factor, mainly they act on the cell membrane phosphatidylcholine of eukaryotic which is found to be a target for phospholipases. These enzymes have a vital role in iron acquisition because of their ability to hemolysis the erythrocytes. Besides these phospholipases are included in epithelial cell invasion and support them to withstand serum [62].

Protein secretion systems
Type II and Type VI protein secretion systems (T2SS & T6SS) give A. baumannii microorganisms the capability of interacting with the host and the environment. Type II protein secretion system uses a two-step pathway to form LipA, LipH, and CpaA effector proteins which are vital virulence enzymes, LipA was declared to possess serine hydrolase activity being capable of hydrolyzing 4-nitrophenyl myristate, also LipA allows the growth of A. baumannii in longchain fatty acids supplemented minimal media as the sole carbon source, revealing its crucial role in nutrient acquisition [63]. LipA and LipH were confirmed as lipases through their ability to cleave para-nitro phenol palmitate. CpaA was previously shown to be a secreted zinc-dependent metalloendopeptidase that was capable of degrading fibrinogen and factor V, deregulating blood coagulation [64].
As well as, T6SS can target other microorganisms through the injection of toxins like peptidoglycan hydrolases, nucleases, and cell membrane toxins and it has a crucial participation in several microbial pathogenesis [12, 65]. Additionally, host nutrient capturing can assist the A. baumannii persistence, the immune system alteration, and dissemination of infection [12,66]. A. baumannii has variable mechanisms for zinc (Zn), manganese (Mn) and iron (Fe)capturing such as Zn-scavenging, the NRAMP (natural resistance-associated macrophage protein) family transporters and siderophores, respectively [12, 66].

A. baumannii Antibiotic Resistance
Various strains of A. baumannii are settlers to variable areas all over the world [67]. MDR strains withstand antimicrobial agents by various resistance mechanisms (Fig. 2); each of them is mainly against certain types of antimicrobial agents.

Cephalosporins
Newly Ambler's class C β-lactamase enzyme showed higher nonsusceptibility toward cefotaxime and ceftazidime than cefepime. It was recovered from a clinical isolate that was found in a Cleveland OH, USA hospital [68]. The phylogenetic analysis of Ambler's class C βlactamase and other A. baumannii, A. pittii, and Oligella urethralis class C β-lactamases, concluded that it was a non-redundant class of class C-β-lactamases. These enzymes were designed as ADC (Acinetobacter-derived cephalosporinases) and this enzyme was assigned as ADC-7, like the other 6 cephalosporinases that had been described previously [68].
A. baumannii Amp C ADC-7 cannot be enhanced by Cefoxitin, in contrast with the majority of chromosomally related class C βlactamases [68]. The insertion of the ISAba1 or ISAba125 sequence to the upstream of the ADC gene increase the ADC expression, resulting in greater promoter activity in comparison with the native promoter activity [69, 70]. Consequently, an increase in the minimum inhibitory concentration (MIC) is required for the various cephalosporins that are affected by these enzymes. Several documents revealed that there are A. baumannii strains that produce extendedspectrum class β-lactamase like ADC-33 provoked nonsusceptibility against cefepime and other cephalosporins [71].

Carbapenems
Carbapenems are the first-line treatment of nonresistant A. baumannii isolates. Although, carbapenem resistance is increasing significantly all over the world and multiple resistance mechanisms are concluded to be involved [

Rifampicin
A. baumannii rifampicin resistance is mainly caused by the alterations of certain β-subunit amino acids of the bacterial RNA polymerase active site. rpo B mutations have also led to elevations in MICs of A. baumannii [91]. In addition, rifampicin enzymatic modification by the ADP ribosyl transferase (ARR-2) and efflux pump activation assist the resistance toward rifampin in A. baumannii strains [92].

Tetracycline
The resistance against tetracyclines mainly tigecycline which has been assigned as one of the "last-resort" antibiotics for the XDR (extended drug resistant) A. baumannii treatment had emerged due to various plasmid-mediated tet(X) gene variants as Tet (X3, X4 and X5), which are mono-oxygenases that can inhibit all tetracyclines including tigecycline and the recently authorized omadacycline and eravacycline [93]. Tetracyclines resistance has been linked to different mechanisms, such as active efflux pumping of the antibiotics to the cell outside through two major efflux systems as the RND (resistance nodulation cell division) family-type pumps especially AdeABC and tetracycline major facilitator superfamily (MFS) efflux pumps as TetA and TetB [94, 95]. Generally, resistance to tetracycline antibiotics had been linked to three major mechanisms: a) ATP-dependent efflux pumps, b) tetracyclines enzyme inactivation, and c) RPPs (ribosomal protection proteins) [96].

Novel treatment options
Treatment varieties for A. baumannii diseases are limited because of the recurrent resistance rate of antimicrobial agents. Especially, the ACB complex which is accompanied by about eighty percent of infections and has provoked nonsusceptibility to all antibiotics that target gram-negative micro-organisms [101]. The disseminated prevalence of A. baumannii antimicrobial resistance, mainly toward lastresort antibiotics, is a crucial alarm. So, the researchers have to pay attention to novel treatment strategies development, some are summarized in Fig. 3.   Fig. 3. Novel therapeutic treatment for A. baumannii infections; TPGS, tocopherol polyethylene glycol succinate, NLTA capped silver nanoparticles and copper nanoparticles capped with N-lauryltyramine.

Combination antimicrobial therapy
A combination antibiotics treatment strategy appears to have the most preferable outcomes for infections compared to monotherapy. Mostly, combinations are depending on polymyxins alongside other antimicrobials that act on the cell wall like carbapenems, vancomycin, and sulbactam. It is not preferable to use polymyxins alone toward infections by high-resistance strains, as this can enhance resistance against them, polymyxins-depending combinations are a suitable alternative to avoid treatment failure. Additionally, tigecycline-depending combinations seem to be efficient toward pandrug-resistant (PDR) strains [102].

Drug repurposing
Major advantages of drug repurposing, are rapidness, cost-effectiveness, and attractive proposition for the treatment of resistant infections. Three repurposed drugs have been suggested by several studies to be promising agents for the MDR A. baumannii infections treatment including, apramycin which is a monosubstituted deoxystreptamine, this distinctive structure that enables the apramycin molecule to intrinsically adapt to almost all resistance mechanisms found in MDR and XDR Gram-negative bacteria [

Bacteriophage therapy
One of the most promising antibacterial approaches is the use of viruses to cure bacterial infections (Bacteriophage therapy), which cures and prevents infectious disorders by bacterial cell lysis. The major benefits of bacteriophage therapy are that the phage specifically assigns the pathological agents instead of the normal cells or the commensals [110]. The initial study concluded that the bacteriophage treatment is effective on A. baumannii was conducted in 2010 [111].
Consequently, the bacteriophage\ antimicrobial combination can be a successful option for the A. baumannii infections therapy, as well as the use of phage\ antibiotic combinations can help to prevent the phage resistance emergency. Some studies have introduced KARL-1 as a promising element combined mainly with other antimicrobial agents for the MDR A. baumannii therapy [112]. A recurrent study showed that a CPS depolymerase (capsular polysaccharide), which is extracted from the TSP (tail spike protein) of φAB6 phage has a major role in inhibiting biofilm development and microorganism attachment to the surface of hospital equipment, also has antimicrobial activity that could destroy microbial cells through membrane lysis [113]. However, this approach has several limitations because most clinical infections are usually due to a variety of pathogenic bacteria, which limits the effect of specific bacteriophages to have a required therapeutic effect [114]. Bacteriophages can also transmit antibiotic resistance genes and toxins to bacteria in the lysogenic state as the bacteriophage genome integrates into the bacterial chromosomal DNA for replication or replicate in a free plasmid-like state [115]. The evaluation of phage therapy curative effects and quality is difficult as it has a more complex composition which includes both proteins and nucleic acids than other protein drugs whose purity and activity can be assessed based on specific antibody titers [116]. The clinical application of phage therapy lacks regulations and policies, so if there is an appropriate regulatory standard it can create opportunities to raise awareness of this promising treatment [117]. Additionally, there is no obvious standard for phage purification and isolation, making the isolated phage preparations efficacy variable. Also, there is no standardized procedure in clinical treatments with bacteriophages [118].

Antimicrobial peptides (AMPs)
Another promising strategy against A. baumannii includes antimicrobial peptides (AMPs) that are a constituent of the nonspecific immunity of eukaryotic and prokaryotic organisms. They seek the attention of the research community because of their broadspectrum activity and low resistance rates [119]. The cationic nature of AMPs allows them to target the cell membranes and disintegrate the lipid bilayer, leading to cytoplasmic leakage and microbial eradication [120]. They have variable amino acids structure with varying amino acids number "from five to > 100" and show a broad spectrum of activity, covering from viruses to parasites [120]. AMPs have other antimicrobial mechanisms including; membrane proteins delocalization [121], and cytoplasmic membrane septum formation alteration [122], they also inhibit the synthesis of the cell wall, proteins, DNA, and RNA [123,124], and have enzymatic activity [125]. Some can eradicate the biofilmforming bacteria by direct biofilm formation inhibition or killing the microorganism inside the biofilm [126]. An updated mouse model experiment showed that the AMP candidates, dN4 and dC4 are efficient in the treatment of pneumonia produced by A. baumannii by inhibiting the biofilm formation in a dosedependent manner and eliminating the established biofilm [127]. But also, the medical applications of antimicrobial peptides have major disadvantages such as high expenses, increased cell toxicity, harsh environmental conditions sensitivity as extreme pH, and instability in the circulatory system [128,129]. However, amino acids in nature allow their structures to be modified easily to decrease their host cell toxicity and protease instability [130].

Human monoclonal antibodies (Hu-mAbs)
Enhancing innate immunity is a promising strategy to eradicate A. baumannii virulent strains, as they can evade the innate immune system, resulting in sustained TLR4 ligation by their LPS which ultimately results in death by sepsis [131,132]. Therefore, many researchers had paid great attention to monoclonal antibodies as a passive immunotherapeutic approach [133]. Human monoclonal antibodies (Hu-mAbs) are a promising strategy for A. baumannii infections treatment, as they are specific and do not affect the commensal microbiota. On the other hand, the opportunity for resistance decline as Hu-mAbs often affect the virulence proteins rather than the survival ones

Nanoparticles
Nanoparticles have attracted much attention as an alternative treatment. Several studies declared the crucial effect of nanoparticles as antivirulence entities for A. baumannii. The TPGS/AgNPs (TPGS-capped AgNPs) hollow structure allows a conformation cavity to load the antimicrobial agents, also its small size allows them to penetrate the bacterial cell wall and cross into bacteria, increasing the antibiotic delivery inside the bacterial cells [137, 138]. Another two studies concluded the efflux inhibitory potential of nanoparticles in A. baumannii as copper nanoparticles capped with NLTA (N-lauryl tyramine) and TPGS (tocopherol polyethylene glycol succinate) capped silver nanoparticles [139, 140]. Additionally, silver nanoparticles have been declared to be capable of suppressing the transcription of various virulence genes and interfering with A. baumannii biofilm production [139]. This delivery system has some limitations including its entrapment in the mononuclear phagocytic system as in the spleen and liver [141]. The nanoparticles drug delivery system reduces the toxicity of the whole formulation, however, the toxicity of the nanoparticles itself is usually studied inadequately. Therefore, proper emphasis on the toxicity of the empty nanoparticles must be taken into consideration, especially in slow or non-degradable particles which can persist and accumulate on the drug delivery site, leading to chronic inflammatory reactions [142].

Targeting OMPA as a potential virulent factor in A. baumannii
Mostly, there are two major strategies to develop a novel antimicrobial agent. One is by inhibiting the essential elements production which helps in the bacterial cell survival [143, 144]; the second is by inhibiting the antibiotic resistance genes or virulence factors to suppress pathogenicity or improve their sensitivity to antimicrobial agents [145]. Although, inhibiting a single essential component inevitably leads to the development of high-level drug-resistant strains [146]. As a result, the novel intervention strategies concentrate on the nonessential processes as a key to overcoming bacterial resistance [147]. There are various strategies to target OMPA including; a synthetic small polypeptide that specifically binds to and blocks OmpA as AOA-2 (a cyclic hexapeptide). AOA-2 blocks the OmpA "without bactericidal activity" decreasing the adhesion of Pseudomonas aeruginosa, A. baumannii, and E. coli to the biotic and abiotic surfaces, and significantly enhancing the sensitivity of A. baumannii [148,149]

Gene editing
Gene editing as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) systems is considered a promising novel strategy for several pathogens treatment. CRISPR-Cas acts by targeting and knocking out antibioticresistance genes and virulence factors. But, a limited number of studies have been conducted on A. baumannii treatment using a gene editing strategy so another research is required [154].

Conclusions
The capability of A. baumannii has an infinite ability to acquire resistance mechanisms, enabling it to persist through the infection pathway and also to survive on healthcare facilities surfaces. However, the major crucial interest is the resistance emergence against the last resort of antimicrobial agents such as colistin and tigecycline which has led to an important challenge in the health care systems. Novel therapeutic options are a must to face the current multi or extended or pan-drug resistant pathogens, especially after the warning alarms of resistance toward "last-resort" antimicrobial agents. COVID-19 alarms the whole world to avoid the intractable nature of infections that can have serious and irreversible social and economic circumstances. The irrational use of antibiotics in hospitals can incredibly result in health hazards. The creation and construction of national and international protocols for antimicrobial stewardship programs would be a beneficial and promising opportunity to help physicians in making evidence-based treatment alternatives concerning antimicrobial treatment and implementation of better antibiotic use in hospitals.

Competing interests
The authors declare that they have no competing interests.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions
Riham A. EL-Hakeem has collected the data for the manuscript under the supervision and guidance of authors; Sarra E Saleh, Mohammad M Aboulwafa, and Nadia A Hassouna, who have written the first draft of the manuscript. Sarra E Saleh and Mohammad M Aboulwafa have helped in writing and revising this manuscript. All authors have read and approved the final manuscript.   Tetracyclines, molecular and clinical aspects.