Microbiological Research 247 (2021) 126722

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

An overview of Acinetobacter baumannii pathogenesis: Motility, adherence

and biofilm formation
Hing Jian Mea a, Phelim Voon Chen Yong a, Eng Hwa Wong b, *
a
School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500, Subang Jaya, Selangor Darul Ehsan, Malaysia
b
School of Medicine, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500, Subang Jaya, Selangor Darul Ehsan, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: The Gram-negative opportunistic pathogen Acinetobacter baumannii has gain notoriety in recent decades, pri­
Acinetobacter baumannii marily due to its propensity to cause nosocomial infections in critically ill patients. Its global spread, multi-drug
Virulence resistance features and plethora of virulence factors make it a serious threat to public health worldwide. Though
Bacterial pathogenesis
much effort has been expended in uncovering its successes, it continues to confound researchers due to its highly
Surface structures
Host-pathogen interaction
adaptive nature, mutating to meet the needs of a given environment. Its persistence in the clinical setting allows
Animal models it to be in close proximity to a potential host, where contact can be made facilitating infection and colonization.
In this article, we aim to provide a current overview of the bacterial virulence factors, specifically focusing on
factors involved in the initial stages of infection, highlighting the role of adaptation facilitated by two-component
systems and biofilm formation. Finally, the study of host-pathogen interactions using available animal models,
their suitability, notable findings and some perspectives moving forward are also discussed.

1. Introduction With such a reputation, it is no surprise that major global health

The rise of Acinetobacter spp, specifically A. baumannii as a nosoco­
mial pathogen has stunned the world and poses a significant threat to
the global health care system. Initially regarded as a low-grade path­
ogen, it is now recognized as an opportunistic pathogen, predominantly
infecting hospital patients making it a leading nosocomial pathogen
with broad clinical manifestations. Most notably in ventilator-associated
pneumonia, blood stream infections, urinary tract infections and men­
ingitis (Bergogne-Berezin and Towner 1996; Peleg et al., 2008). Earlier
cases can be traced back to the early 2000s as reports began to surface
from wounded veterans and soldiers who served in Iraq and Afghanistan
repatriated to be treated for conflict induced trauma contracting the
infection (Peleg et al., 2008). So common were instances of infection
from Acinetobacter spp., with cases as high as 33 % of all wound and
blood isolates, that it garnered the nickname ‘Iraqibacter’ (Petersen
et al., 2007). Since then, outbreaks have been reported in all major
continents (except for Antarctica), with cases cause by multi-drug
resistant isolates reaching as high as 44 %. This has led to poor pa­
tient prognosis which further strains an already struggling global health
care infrastructure as our existing antimicrobial solutions are rendered
ineffective (Dijkshoorn et al., 2007; Giammanco et al., 2017).
authorities including the European Centre for Disease Prevention and
Control (ECDC), Infectious Diseases Society of America (IDSA), WHO
and Center for Disease Control and Prevention of America (CDC) have
declared multi-drug resistant A. baumannii a critical threat to global
health, with the latter promoting Carbapenem-Resistant Acinetobacter
from a ‘serious’ threat level to ‘urgent’ in 2019 (CDC, 2019; Pendleton
et al., 2013; Rello et al., 2018; Tacconelli et al., 2017). It has also been
considered the most serious among the ‘ESKAPE’ organisms (Entero­
coccus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. bau­
mannii, Pseudomonas aeruginosa, and Enterobacter species), so named due
to their proficient ability to escape the effects of antimicrobial drugs
(Boucher et al., 2009). Furthermore, the WHO has in a recent publica­
tion considered carbapenem-resistant A. baumannii a priority 1 critical
bacteria in their list of bacteria, outscoring even the 2nd priority bac­
teria in terms of ranking by 9.3 % within their own predetermined
criteria (Tacconelli et al., 2018).
Collectively, much attention has been directed on studying clinically
relevant A. baumannii, with attention to its virulence and pathogenesis.
These studies have illuminated a great deal of knowledge in under­
standing the pathogen and what makes it so ‘successful’ with its diverse
repertoire of virulence factors, providing significant insights aimed at

* Corresponding author.
E-mail address: EngHwa.Wong@taylors.edu.my (E.H. Wong).

https://doi.org/10.1016/j.micres.2021.126722
Received 24 September 2020; Received in revised form 29 January 2021; Accepted 1 February 2021
Available online 4 February 2021
0944-5013/© 2021 Elsevier GmbH. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
H.J. Mea et al. Microbiological Research 247 (2021) 126722

exposing possible weaknesses and targets for the development of novel Table 1
treatment and preventive strategies. Special interest has also been given Identified list of virulence factors along with their roles in pathogenesis for
in understanding the pathogen’s ability to survive, persist and thrive in Acinetobacter baumannii.
diverse environmental conditions especially in clinical settings which is Virulence factor Proposed role in References
believed to be directly linked to its development of multi-drug resistance pathogenesis
and hospital outbreaks. This article seeks to summarize the current Pili Motility, adherence, Piepenbrink et al., 2016;
characterization of virulence factors (Table 1) contributing to patho­ biofilm development Tomaras et al., 2003
genesis focusing on its ability to move, adapt by adhering/attaching and Porin (OmpA, Omp33-36, Porins (OmpA, Omp33- Choi et al., 2005, 2007,
Omp22, CarO, OprD- 36, Omp22, CarO, 2008; Gaddy et al. 2009;
colonize any given environment via forming biofilm along with its reg­
like) OprD-like), host cell Kim et al., 2016; Kwon
ulatory mechanisms and current means of studying the complex host- death et al., 2019; Lee et al.,
pathogen interaction during the initial stages of infection. Special 2001; Smani et al.,
attention is also given to surface related features and proteins that 2012a, 2013a; Rumbo
contribute to its pathogenesis. et al., 2014.
Biofilm associated proteins Adherence; biofilm Brossard and
(BAP) development and Campagnari, 2011;
2. Virulence factors maturation Loehfelm et al., 2008
BAP like Proteins (BLP) Adherence De Gregorio et al., 2015
In the past decade, many have undertaken the task to unravel the Lipopolysaccharide (LPS) Adherence, Serum Bartholomew et al.,
resistance, survival in 2019; McQueary et al.,
unique and complex mechanisms that contribute to the successful
tissue infection, evasion 2012
emergence of A. baumannii to the clinical scene. Interestingly, most of the host immune
nosocomial pathogens like E. coli, P. Aeruginosa and S. Aureus have response
distinct molecular determinant or toxins primarily responsible for Penicillin-binding protein Adherence, serum Lee et al., 2008
causing disease. This has not been the case for A. baumannii, instead 7/8 and b-lactamase resistance, in vivo
PER-1 survival
bacterial pathogenesis has been attributed to a combination of factors Trimeric Autotransporter Adherence, biofilm Bentancor et al., 2012;
and an arsenal of virulence factors working in concert to cause an (Ata) development Thibau et al., 2019;
infection (Fig. 1). It begins with the surface of the bacterial cells which Weidensdorfer et al.,
being made up of a plethora of molecular components interacts with the 2016
FhaBC secretion system Adherence, host cell Pérez et al., 2016
environment. The current known list of virulence factors identified in
death
A. baumannii are summarized in Table 1. Capsular polysaccharide Adherence, growth in Russo et al., 2010;
serum, survival in tissue Lees-Miller et al., 2013
3. Bacterial pathogenesis infection, biofilm
development
Phospholipase (PLC and Serum resistance, Fiester et al., 2016
3.1. Initial stage of infection PLD) invasion, in vivo survival
Outer membrane vesicle Delivery of virulence (Jin et al., 2011).,Jun
For a bacterium to become pathogenic, contact must first be estab­ (OMV) factors, horizontal et al., 2013
lished with a given host surface through adherence before expressing transfer of resistance
gene
virulence factors for colonization (Falagas and Rafailidis, 2007). Here
Iron acquisition system In vivo survival, Fiester et al., 2016;
they transition from a planktonic free-swimming mode of growth to that (acinetobactin and NfuA) persistence, host cell Gaddy et al., 2012
of a surface-orientated one. It has been recognized that one of the major death
contributing factor to the rise of nosocomial A. baumannii stems from its Zinc acquisition system In vivo survival, Lonergan et al., 2019
ability to adhere and colonize biotic and abiotic surfaces, employing a (ZnuABC, ZigA & ZrlA) persistence
Manganese acquisition In vivo survival, Green et al., 2019
change of strategy currently dubbed ‘persist and resist’ rather than the
system (MumC and persistence, immune
traditional toxin expression of other pathogens. This, coupled with an MumT) evasion
equally confounding capacity to survive in normally unfavorable con­ Type II protein secretion In vivo survival Harding et al., 2016
ditions makes A. baumannii a formidable pathogen. system
Type VI protein secretion Killing of competing Repizo et al., 2015;
system bacteria, host Silverman et al., 2012
3.2. In search of a target - motility colonization
Tuf (plasminogen binding Immune evasion, Koenigs et al., 2015
Normally, bacterial cells exist in a planktonic free moving state. Here protein) complement system
they employ a variety of techniques to propel themselves around. It is supression
RecA Motility, In vivo Aranda et al., 2011;
ironic for Acinetobacter spp. to have received such a name as the original survival, biofilm Corral et al., 2020a
Greek word from which it is derived translates to mean nonmotile rod. development
This was initially proposed back in 1954 by Brisou and Prevot, 1954 who GigABCD In vivo survival, killing Gebhardt and Shuman,
attempted to distinguish the organisms based on its motility. This of host cells 2017
AdeRS Antimicrobial Montaña et al., 2015;
however is not the case for the two clinically relevant Acinetobacter
resistance, virulence Richmond et al., 2016;
species, A. baumannii and A. nosocomialis which have been demonstrated regulator Sun et al., 2017
to possess both surface-associated and twitching motility (Eijkelkamp BaeSR Antimicrobial Lin et al., 2014
et al., 2011a; Skiebe et al., 2011). The direct correlation between resistance, virulence
motility and ability to cause disease during the initial phase of infection regulator
BfmRS Csu Pili expression; Kim et al., 2019; Liou
has been established in other pathogens such as E. coli, Salmonella spp. virulence regulator et al., 2014
and P. aeruginosa (Josenhans and Suerbaum, 2002; Kao et al., 2014). CheAY Csu Pili expression; (Chen et al., 2017)
However, unlike other bacteria, A. baumannii along with other species of virulence regulator
Acinetobacter have been shown to lack flagella thus making the link GacAS and PaaE (two Neutrophil recruitment, Cerqueira et al., 2014;
component system & virulence regulator Bhuiyan et al., 2016
between motility and virulence difficult to establish.
phenylacetic acid
Twitching motility is a kind of jerky movement in a wet surface that metabolism)
is flagella-independent and has been characterized and studied in other (continued on next page)
genera like Pseudomonas, Neisseria, Myxococcus and Ralstonia (Corral

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H.J. Mea et al. Microbiological Research 247 (2021) 126722

Table 1 (continued )
Virulence factor Proposed role in References
pathogenesis
OmpR/EnvZ Killing of host cells, (Tipton and Rather,
virulence regulator 2017)
PmrAB Antimicrobial Beceiro et al., 2011; Chin
resistance, LPS et al., 2015
modification
et al., 2020a, 2020b; Mattick, 2002; Talà et al., 2019). It involves the use
of a functional type IV pili (TFP) that cycles between repeated rounds of
extension and retraction that pulls the bacterial cells forward (Jarrell
and McBride, 2008; Mattick, 2002). Genes that have been identified to
be involved in pili assembly are pilA, pilB, pilC, pilF, pilM, pilN, pilO, pilP,
pilQ, pilZ and pilW, along with genes involved in twitching pilR, pilS, pilT
and pilU (Clemmer et al., 2011). Several studies have shown that most of
the genes mentioned are present across different clonal groups of clin­
ically A. baumannii strains and show high levels of conservation, sug­
gesting a common feature among the species including the reference
strain ATCC® 17978™ & 19606™ (Eijkelkamp et al., 2011a, 2011b;
Eijkelkamp et al., 2014; Piepenbrink et al., 2016). However, high vari­
ations have been recorded primarily in the fimbrial protein PilA which is
a major pilin subunit thought to contribute to the mechanism allowing
for host immune system evasion. This variation is also observed when
comparing within genera between A. baumannii and A. nosocomialis,
with surprising similarity among other bacterial species compared,
implying that functional requirements determine the protein structure
(Piepenbrink et al., 2016; Ronish et al., 2018). The same study also
attributed TFP to promoting host cell adherence, which will be further
discussed in the next section.
An alternative form of motility observed in A. baumannii clinical
isolates is called surface associated motility seen among bacteria on
semi-solid surfaces. It is sometimes termed as ‘swarming’ motility and
was first characterize in Acinetobacter isolates by Barker and Maxted
(1975). Initially thought to be directed by the type IV pili, a study by
Clemmer et al. (2011) showed that the loss of the pilT gene resulted in 54
% motility reduction, with the residual being attributed to an alternative
form of motility. Another study by Wilharm et al. (2013) corroborated
this finding and added that pilT inactivation can interfere but not abolish
surface motility using a transformed mutant strain. Additionally, LPS
and K1 capsule have also been implicated to have a role in
surface-associated motility though more work could be done in vali­
dating this (McQueary et al., 2012). In an attempt to characterize a
larger sample group, Skiebe et al. (2012) found that among 83 different
A. baumannii isolates all but one exhibited motility on a semi-solid
surface of 0.5 % agarose. This was challenged by other studies which
contradicted this claim (Clemmer et al., 2011; Eijkelkamp et al., 2011b),
although it should be noted the experimental conditions differ with one
study by Dahdouh et al. (2018) showing low surfaced associated motility
among Spanish isolates but high motility in Lebanese isolates. These
studies serve to highlight the need for a comprehensive and represen­
tative study to characterize a large sample of A. baumannii clinical iso­
lates which should aim at correlating virulence and motility.
Interestingly, several studies have shown that a loss of motility

Fig. 1. A schematic diagram depicting the bacterial cell envelope and surface related virulence factors involved primarily in motility, adherence and biofilm for­
mation. These include Lipooligosaccharides & Lipopolysaccharides (LOS & LOS), Outer membrane protein A, Csu Pili, Type 4 pili (TFP), Biofilm associated protein
(BAP) and trimeric auto transporter (Ata).

3
H.J. Mea et al.
resulted in a significant attenuation of the pathogen. An example of this
was found when two immotile mutant isolates were found to have had
their motility restored via addition of 1, 3-diaminopropane (DAP) in the
growth media. The mutants were found to have mutation transposon
insertions in dat and ddc genes preventing the biogenesis of DAP which
also resulted in A. baumannii attenuation in a Galleria mellonella cater­
pillar infection model (Skiebe et al., 2012). Another example is by
Pérez-Varela et al. (2017) where an ATCC® 17978™ mutant harboring
an amino acid substitution in position 520 or 540 of the RpoB protein
that conferred rifampin resistance was found to have impaired
surface-associated motility which also affected the pathogen’s virulence
capabilities in a C. elegans infection model. Other factors that have been
shown to affect motility include two-component system, quorum
sensing, light, salinity, iron and lipopolysaccharides demonstrating the
close correlation between motility and extracellular conditions (Eijkel­
kamp et al., 2014; McQuery et al. 2012; Pérez-Varela et al., 2017; Skiebe
et al., 2012; Wood et al., 2018)
Taken together this presents a promising route of study in the
development of novel therapeutic strategies specifically targeting
motility against A. baumannii and may have applications for the entire
genus as this trait seems to be consistent in other species. There are
however several challenges with this, one of which have already been
highlighted above with regards to the type IV pili where the major pilin
subunit possesses high variations seen in both amino acid sequence and
glycosylation patterns (Ronish et al., 2018). Another would be
phase-variation first reported in 2015 which needs to be accounted for
when studying bacterial virulence which could lead to conflicting results
and ineffective therapeutic strategies prompting the need to revisit all
current A. baumannii studies to ascertain certainty (Ahmad et al., 2019;
Tipton, Dimitrova and Rather, 2015).
3.3. Surface-related features
Before adherence can take place the repulsive forces that exist be­
tween the surfaces of both bacterial and host target cells due to it being
negatively charged must be overcome. This is thought to be accomplish
mainly by establishing initially weak hydrophobic interactions that are
reversible before more permanent fixtures are established (Rosenberg
et al., 2013). Cell Surface hydrophobicity (CSH) is known to be an
important parameter that enables microorganisms to attach to hydro­
carbon found on surfaces and or cells, moving from water to organic
phases in a given environment which aides in the organisms survival as
it seeks for carbon sources (Krasowska and Sigler, 2014). Surfaces which
are hydrophobic will give rise to adherence from hydrophobic cells and
vice versa for hydrophilic surfaces. It should also be noted that micro­
organisms have the capacity to manipulate their cell surface hydro­
phobicity depending on the environmental conditions and growth
phases that they are in although this has yet to be confirm for Acineto­
bacter spp. (khelissa et al., 2017; Vij et al., 2019). This is an important
area of concern when considering how the plethora of medical devices
and implants such as catheters, mechanical heart valves and respiratory
tubes are made of hydrophobic materials and have been shown to harbor
microbe presence including biofilm-forming A. baumannii (Greene et al.,
2016a). Links between bacterial CSH affecting pathogenicity and sub­
sequently virulence have been studied among various microorganisms
with results varied showing that it may positively or negatively modu­
late virulence. Pertaining to A. baumannii, a previous study has shown
that CSH correlated with increased adherence to abiotic surfaces which
then leads to biofilm formation explaining bacterial clinical persistence
(Pour et al., 2011). In addition, a recent study which analyzed 36 clinical
strains of A. baumannii from both International Clone Lineage I and II (IC
I & IC II) found IC I strains to be more hydrophobic than IC II in general
leading to the formation of conventional biofilm and adherence to
abiotic (plastic) surfaces. This observation was also true when
comparing within IC II clonal lineage cells with more hydrophobic IC II
strains displaying better biofilm and adherence capacity than
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Microbiological Research 247 (2021) 126722
hydrophilic strains. Interestingly, hydrophilic IC II strains adhered bet­
ter to lung epithelial cells and were more virulent in C. elegans and
murine infection model (Skerniškytė et al., 2019). One study also found
that a mutant A. baumannii strain that lacked a redox protein thioredoxin
A was more hydrophobic making it more susceptible to J774 macro­
phage uptake which points to better bacterial clearance, although this
was not tested in an in vivo model (May et al., 2019). Both studies do
however suggest that explorations into manipulating cell surface hy­
drophobicity could lead to uncovering novel therapeutic strategies to
combat nosocomial A. baumannii infections highlighting another aspect
of its pathogenesis.
3.4. Attachment and the initiation of infection
3.4.1. Attachment/adherence
In its fight for survival during this critical moment, A. baumannii
must decide what to do next whether to remain in a free moving state or
attach to a surface and start colonizing it. Usually, a major determining
factor is nutrient availability particularly low nutrient availability. This
sounds counter intuitive, however bacteria grown in high nutrient en­
vironments either fail to form biofilm of form loose structures that are
easily disrupted by fluid shear (Petrova and Sauer, 2012). This is
consistent for A. baumannii as a study found that under iron limiting
conditions one of the most notable changes other than slow growth rates
was a reduction in motility specifically via the T4P and type I pili (Eij­
kelkamp et al., 2011b). A comprehensive transcriptome analysis
compared mRNA expression profiles of biofilm and planktonic cells
found that there was a down-regulation in genes associated with motility
and an upregulation of genes associated with biofilm formation. These
same genes contribute to attachment and adherence as the first step in
establishing a mature biofilm (Rumbo-Feal et al., 2013). A combination
of these 2 approaches applying transcriptome profiles on a wide popu­
lation of A. baumannii strain would validate these correlations especially
in the utilization of model strains representative of the current clinical
context. Collectively, these studies highlight the sequence of events that
are essential for the pathogen to begin colonization starting with
attachment. It should be noted that there have emerge some reports that
challenged this claim with clinically isolated A. baumannii lacking the
ability to adhere to A549 cells in an in-vitro study (Lázaro-Díez et al.,
2016). Hence, existing evidence should be viewed with caution, given
the limitations of these studies which only serve to highlight the need for
a consistent model for virulence and pathogenicity studies on
A. baumannii.
An early study on the subject found that A. baumannii attachment to
abiotic surfaces such as plastic and glass was mediated by a pili assembly
that was visualized under electron microscopy. Thorough investigation
identified these pili to be the product of a chaperone-usher pili assembly
that resembled that of other bacteria later named CsuA/BABCDE-
mediated pilus. This pilus has been found to be widely conserved and
common in nearly all A. baumannii clinical isolates (Moriel et al., 2013;
Tomaras et al., 2003). The CsuA/BABCDE-mediated pilus consist of two
subassemblies, a short thin tip fibrillum encoded by the csuE gene that is
mounted on second subassembly helical rod encoded by the csuA/B gene
(Pakharukova et al., 2017; Tomaras et al., 2003). They function as a
surface recognition adhesion site and pili biogenesis respectively and are
very much dependent on environmental conditions to regulate expres­
sion (Hospenthal et al., 2017). Csu functional studies revealed that
absence of CsuA/BABCDE-mediated pilus production resulted in the
lack of attachment and biofilm-forming capacity of the mutant strain to
plastic. However, attachment was still present on eukaryotic cells,
mainly surfaces of fungal filaments and A549 cells (Gaddy et al., 2009).
Later studies however found that the CsuA/BABCDE-mediated pilus is
not involved in the adherence and attachment of A. baumannii to host
cells, in this case bronchial epithelial cells but is essential for biofilm
formation on abiotic surfaces (de Breij et al., 2010; Lee et al., 2006).
Therefore, the mechanism of attachment to host cells are independent of
H.J. Mea et al.
CsuA/BABCDE-mediated pilus. For a more comprehensive review of the
pilus biogenesis in Gram-negative bacteria the reader is referred to the
following article (Hospenthal et al., 2017).
A. baumannii has also been found to produce biofilm-associated
protein (Bapab) which is part of the biofilm forming and maturation
process first characterized in S. aureus. It is also discovered to play a
critical role in adherence (Brossard and Campagnari, 2011; Loehfelm
et al., 2008). Initial sequence analysis revealed a repetitive structure
consistent with bacterial cell surface adhesins which are large surface
proteins possessing core domain tandem repeats. Most identified Aci­
netobacter genus homologues have a 80–100 aa long Ig-like repeats
which share structural and functional similarities but not similar pri­
mary sequence, hence displaying clear heterogeneity (De Gregorio et al.,
2015). This protein was found to be prevalent in most A. baumannii
strains, with one study reporting presence in all clinically relevant
strains along with an avirulent strain with the only exception from a
community acquired strain (Eijkelkamp et al., 2014; Yang et al., 2019).
Similarly, all but one isolate from an Australian Hospital spanning a
decade were found to possess the bap gene (Goh et al., 2013). Bap has
been found to modulate the cell surface hydrophobicity by increasing it,
which results in improve observed adherence to epithelial cells (Bros­
sard and Campagnari, 2011). Recently, two additional proteins were
discovered to share a large sequence motif at the NH2 terminus with BAP
subsequently called Bap-like proteins, designated BLP1 & BLP2. In one
study, BLP1 has been found to be unevenly distributed in A. baumannii
but present in other Acinetobacter spp. with a large sequence from 3044
up to 3356 aa. Whereas BLP2 is more conserve in A. baumannii and
found in all ACB complexes tested with a smaller size of 728aa protein.
Both proteins are co-expressed with BAP suggesting complimentary
functions with significant drop in adherence to A549 cells observed in
Δblp1 and Δblp2 mutant (De Gregorio et al., 2015).
In addition, other notable factors involved in attachment and
adherence of A. baumannii is the expression of blaPER-1 a broad range
β-lactamase gene (Lee et al., 2008). The extent of involvement of this
gene with adherence is still not fully established as studies regarding its
distribution among clinical isolates found that not all strains harbor the
gene. Although initially thought to contribute to biofilm formation, its
positive correlations through prevalence studies have been under­
whelming (Bardbari et al., 2017; Yang et al., 2019). The Acinetobacter
trimeric autotransporter adhesin (Ata) first identified in A. baumannii
ATCC® 17878™ was found to mediate bacterial adhesion to extracel­
lular matrix proteins and forms an important virulence factor in most
Gram-negative bacterium (Bentancor et al., 2012; Weidensdorfer et al.,
2016). Ata deleted mutants were found to be significantly attenuated in
Galleria mellonella larvae models as adhesion was completely abolished.
In addition, Ata was found to induce strong immunogenic response
demonstrating its critical role in the infection process and potential use
as a target for vaccine and therapeutic development (Thibau et al., 2019;
Weidensdorfer et al., 2016). Some notable mentions include fibronectin,
poly-β-1,6-N-acetylglucosamine (PNAG) and porins which will be dis­
cussed below. It is also worth mentioning that many of the components
brought up thus far play multifaceted roles especially in initiating and
supporting biofilm growth and maturation which is integral to
A. baumannii’s pathogenesis to successfully colonize.
3.4.2. Porins/OMP
Another notable feature of A. baumannii are the outer membrane
proteins (OMP) that litter the surface of the outer membrane (OM).
Surrounded by two concentric lipid bilayer membranes, gram negative
bacteria have a lipid component of the inner cytoplasmic membrane
composed of phospholipids distributed across the inner and outer leaf­
lets (Fig. 1). The inner leaflet has a composition that is similar to the
cytoplasmic membrane. In contrast, the outer leaflet is asymmetric
being made up of mainly lipopolysaccharides (LPS) (or lip­
ooligosaccharides (LOS) in the case of A. baumannii due to the absence of
o-polysaccharides) on the outer portion and lipid bilayer in the inner
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Microbiological Research 247 (2021) 126722
portion (Bartholomew et al., 2019). Functionally, the outer membrane
allows for more transmembrane transport across compared to the
cytoplasmic membrane. This is mainly facilitated by porins which can
make up about 50 % of the outer membrane. Porins in A. baumannii also
serve similar functions mainly controlling the movement of solutes
across the OM but have also been shown to contribute to antimicrobial
resistance and as a virulence factor which is common among most
gram-negative bacteria.
The first porin to be identified and characterize is OmpA (first
identified as Omp 38). It is a β-barrel porin with a 2-nm pore diameter
and a C-terminal periplasmic globular extension which accommodates
molecules up to 500 Da in size (Sugawara and Nikaido, 2012). It is
highly conserved among bacterial species and has also been found in
A. nosocomialis, which is another clinically relevant species of the family
with a 92 % amino acid sequence similarity (Kim et al., 2016). This
protein has been found to have low permeability when compared with
similarly sized porins of other bacteria (e.g. OprF of E. coli) thus impli­
cating it in antibiotic resistance (Kwon et al., 2019; Sugawara and
Nikaido, 2012). OmpA has also been discovered to contribute to bac­
terial virulence by adhering to host lung epithelial cells through direct
interactions with fibronectin on cell surfaces (Gaddy et al., 2009; Smani
et al., 2012a, 2012b). This induces an inflammatory response which
could also lead to cellular invasion and subsequent host cell death
through the activation of caspase-3 as OmpA has been detected to
localize in both mitochondria and nuclei (Choi et al., 2005, 2007; 2008;
Lee et al., 2001). In addition, OmpA has been found to constitute a large
abundance in these outer membrane vesicles (OMV) secreted by
A. baumannii, conferring upon it cytotoxic effects (Jin et al., 2011). This
is further supported by clinical data that demonstrates high bacterial
OmpA expression directly correlates with increase mortality rates.
Conversely, attenuated phenotype was observed in murine and
G. mellonella models infected with ΔAbOmpA mutant clinical strains
(Sánchez-Encinales et al., 2017).
Other porins more recently discovered include Omp34 (previously
known as omp33-36), which similar to OmpA also contributes to bac­
terial pathogenesis, adhering and inducing lung epithelial cell death via
activation of caspases (Rumbo et al., 2014; Smani et al., 2013). Adher­
ence using Omp34 has also been attributed to be fibronectin-dependent
(Smani et al., 2012b). Another porin CarO, is responsible for carbape­
nem resistance in A. baumannii functions similarly to the other porins
also promoting cell adhesion. It is also capable of manipulating host cell
immune response by reducing NF-κB pathway expressing genes, ach­
ieved through suppressing IκBβ phosphorylation. The exact mechanism
is still uncertain but current observations suggest its role in bacterial
persistence within a given host (Sato et al., 2017; Zhang et al., 2020). To
the best of our knowledge, since most of the studied porins have high
immunogenic, cytotoxic effects and contribute to host cell adherence, it
is no surprise that they have been the subject of intense vaccine target
studies. Suffice to say, porins are a major virulent factor and are thus a
prime target for controlling A. baumannii pathogenesis before and dur­
ing infections. Particular attention could be paid to what governs porin
expression as disruption have been seen to affect outer membrane sta­
bility and reduce antimicrobial resistance (Kwon et al., 2019)
3.5. Two component regulatory systems
Much of the bacterial virulence factors stated in this review and used
by A baumannii are regulated by two-component systems that directly
affect the genetic expression of the pathogen in response to environ­
mental signals. This by itself is a large topic and deserves a separate
review on its own, as such we will only briefly look into some common
ones that directly affect the pathogenesis in the initial phases of infec­
tion. Two component systems (TCS) are ubiquitous signal transduction
systems that consist of sensor kinases embedded in the cytoplasmic
membrane that sense surrounding extracellular and intracellular stimuli
(Groisman, 2016). These sensors usually respond to environmental
H.J. Mea et al.
physiological stimuli like pH, osmotic pressure and envelope stresses
leading to the relaying of the sensor kinase via phosphorylation. This is
followed by a conformational change of the transcription factor which
leads to DNA binding and transcription (Kröger et al., 2017). A feedback
mechanism is vital for the process to function optimally ensuring that
output is adjusted to reflect the organism’s needs at a given situation.
This is also evident in all two component systems which play a role in
not only stress response but, pathogenesis, symbiotic relations with host
cells, antimicrobial synthesis with competing microbes and antibiotic
resistance (Groisman, 2016).
Current studies into A. baumannii TCS have revealed seven systems
that contribute to virulence. These include AdeRS, BaeSR, BfmRS,
CheAY, GacSA, ompR-envZ and PmrAB. AdeRS TCS controls the
expression of three RND efflux pumps AdeABC, AdeFGH and AdeIJK;
which have been characterized to be closely associated with conferring a
multidrug resistant phenotype against aminoglycosides, tetracyclines,
tigecycline, erythromycin, fluroquinones and chloramphenicol (Mon­
taña et al., 2015). Studies have shown that AdeRS deletion resulted in
more than 579 genes differentially expressed. These included genes
related to virulence, most notably a drop in biofilm formation, antimi­
crobial resistance, and host killing capacity during infection (Richmond
et al., 2016; Sun et al., 2017). Similarly, BaeSR also regulates expression
of major efflux pumps, though this has been proposed to be of an indirect
nature (Lin et al., 2014). BfmRS was discovered to affect the expression
of CsuA/BABCDE, thus also affecting morphology, biofilm expression,
attachment to both abiotic and biotic surfaces and microbial resistance.
Disruption of BfmRS prevents host cell colonization and subsequent
infection. Interest into this TCS as a potential target for antimicrobial
treatment has yielded mixed findings. One example is the identification
of the TCS’s role in bacterial capsule production and cell wall homeo­
stasis which when disrupted reduces bacterial survival in human serum
but subsequently increases host cell cytotoxicity due to an increase in
OMV production (Kim et al., 2019; Liou et al., 2014). These studies also
recorded increase bacterial resistance to several antibiotics, hence the
role of BfmRS in modulating multiple virulence factors. CheAY, a more
recently discovered TCS, has been implicated in affecting virulence
through the decrease in homoserine lactone synthase expression. This
results in impaired cell motility and biofilm formation regulated by
CsuA/BABCDE chaperone/usher pili in an AbaI-dependent manner
(Chen et al., 2017).
PmrAB contributes to polymyxin B and colistin-resistance strains not
just for A. baumannii but also in P. aeruginosa and K. pneumoniae (Beceiro
et al., 2011). This is believed to be possible through the regulation of
genes involved in lipopolysaccharide modification, with phosphoetha­
nolamine conferring colistin resistance that’s results from an increase in
bacterial surface charge that repels cationic polymyxins (Beceiro et al.,
2011; Chin et al., 2015). Another more recently discovered TCS of
A. baumannii is the ompR-envZ which regulates porin expression trig­
gered by environmental osmolarity differences. Interestingly, a muta­
tion to the this TCS resulted in decreased G. mellonella virulence and
drastically increased colony morphology switching frequency from
opaque to translucent, which is an indication of phase variation (Tipton
and Rather, 2017). First identified through a mutagenesis in a trans­
poson screen ATCC® strain 19606™, the TCS GacAS sensor kinase GacS
was identified when the mutant was found to be unable to metabolize
citrate as a sole carbon source (Dorsey et al., 2002). Later revealed to be
directly involved in bacterial virulence, GacAS deletion affected more
than 674 genes expression with a wide range of functions, including
motility (specifically type I pili) and the CsuA/BABCDE chaperone-usher
pili assembly system, resulting in attenuated virulence in a mice septi­
cemia model (Cerqueira et al., 2014). Collectively, TCS are vital for
bacterial responses to environmental changes, regulating gene expres­
sion in order to adapt and survive. Although substantial, studies into
identifying similar trends in clinical strains are still needed in order to
validate TCS role in virulence as most of the studies quoted employed
reference ATCC® strains. Targeting and disrupting TCS to consequently
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affect he pathogenesis of A. baumannii and effectively control infection
presents an interesting area for further investigation.
3.6. Biofilm
Upon successful attachment to a host/surface, A. baumannii will
begin to express several virulence factors to colonize this new space by
the formation of a biofilm. Some of those factors have already been
discussed in the previous sections and will be further expounded here as
they also play a significant role in biofilm formation and maturation.
This highly regulated event starts with the bacteria forming tertiary
structures where close contact is made between each bacterial cell to be
encased in an extracellular matrix that comprises of carbohydrates,
proteins, nuclei acids and various other macromolecules (Dufour et al.,
2010). Biofilms provide a means for the bacteria to survive in harsh
environments and have also been associated with the pathogen’s ability
to resist various antimicrobial assaults occurring in three stages; early
development, matrix formation and maturation (Hoyle and Costerton,
1991). Tests have shown that clinically isolated A. baumannii biofilm
forming cells are able to survive desiccation twice as long when
compared to their non-biofilm forming counterparts. However, these
same studies have found that the strong biofilm forming isolates seem to
be less resistant to antimicrobial agents suggesting a certain trade-off
between antibiotic resistance and desiccation tolerance (Espinal, Marti
& Vila 2011; Greene et al., 2016a). This seeming contradiction may be
due to the use of different strains in these studies which highlights the
need to standardize and create a more consistent experimental designed
starting with genetic profiling of the strains used.
Indeed, biofilm production has been observed in A. baumannii and is
considered a major virulence factor that greatly enhances its survival
and persistence. Consequently, A. baumannii biofilm have been found to
be present on a whole host of medical devices such as catheters, endo­
tracheal tubes of respirators, dialysis tube bottles, stainless steel, and
polycarbonate surfaces (Gaddy and Actis 2009; Greene et al., 2016b).
Greene et al. (2016b) showed that among 6 surfaces tested for
A. baumannii biofilm growth, polycarbonate which is a durable, low-cost
plastic favored for its ability to undergo autoclaving was the most ac­
commodating surface for biofilm growth, making it an ideal reservoir.
Additionally, one study found that despite long term desiccation and
starvation up to 60 days, five clinical isolated strains of A. baumannii
were able to maintain virulence on surfaces such as plastic, glass and
even lab coats though at decreases culturability (Chapartegui-González
et al., 2018). With such persistence within a hospital environment, it is
natural for the pathogen to be eventually the cause of numerous hospital
outbreaks documented over the decades (Dijkshoorn et al., 2007; Greene
et al., 2016b).
Although, many studies have suggested that the formation of biofilm
varies from strain to strain, some commonalities have been observed
with most A. baumannii initiating biofilm formation through the
expression of type I chaperone-usher pilus system. Studies have shown
that a large majority of clinical A. baumannii isolates can form biofilms,
which is significantly higher when compared to environmental isolates
(Bardbari et al., 2017; Eijkelkamp et al., 2011a, 2011b). They also
observed that strains from International Clone II usually form larger
biofilm than their counterparts, begging the question of their correlation
to pathogenicity. The Csu pili is interesting as it has dual roles in bac­
terial adherence and biofilm formation. Its deactivation resulted in the
loss of adherence to abiotic surface and biofilm forming capabilities (de
Breij et al., 2009; Pakharukova et al., 2018). This however is not the case
with regards to interactions with biotic surfaces as adherence of Csu
mutants were still observed, suggesting an alternative means of adher­
ence (Gaddy & Actis 2009). This poses a challenge to therapeutic
development aimed against the Csu pili as its usefulness would be
limited to controlling environmental and clinical devices colonization
and not necessarily suited for infection control.
Part of the biofilm initiation strategy once attachment has been
H.J. Mea et al.
established is the formation of extracellular polymeric substance (EPS)
matrix that is composed of polysaccharides, extracellular DNA (eDNA),
lipids and proteins. The EPS forms an irreversible surface attachment
resulting in bacterial persistence (Petrova and Sauer, 2012). This has yet
to be clearly defined and establish in A. baumannii studies. One of the
well documented polysaccharides that is produced by A. baumannii
during this process is poly-β-(1-6)-N-acetylglucosamine (PNAG) which is
common among other bacteria such as E.coli (Choi et al., 2009; Nait
Chabane et al., 2014). Encoded by the pgaABCD gene cluster which
shares close homology with the pga locus of E.coli, PNAG produced by
the bacteria helps form the landscape of the biofilm which allows the
bacteria to attach facilitating surface-to-cell and cell-to-cell adhesion as
well as pellicle formation. Deletion of this gene resulted in bacteria that
could aggregate but not form multilayer three-dimensional structures
(Choi et al., 2009; Nait Chabane et al., 2014). In addition, upregulation
of PNAG expression and its transporter was observed in a colistin
resistant strain which points to it being regulated by the PmrAB two
component system (Park et al., 2015). Another EPS confirmed to be
release by A. baumannii is extracellular DNA (eDNA). Essentially, eDNA
is made up of the genomic DNA of the bacteria and found to be release in
three ways: (i) active release in a free form, (ii) encapsulation by
membrane vesicles and released (early stages of growth), and (iii) pas­
sive release during cell death and lysis (late stages of growth). eDNA
results in biofilm augmentation as its introduction in cell media pro­
duces a larger biomass. Conversely, DNAase I presence leads to
observable reduction in biofilm mass (Sahu et al., 2012). Though an
important component of biofilm formation, it is unclear precisely how
does eDNA contribute or regulate this process. With reference to
P. aeruginosa, eDNA was observed to act as a scaffold, intracellular
connector and molecular linker due to it being polyionic (Whitchurch
et al., 2002; Yang et al., 2007). Similar connections could be made that
require further investigations into the role of the various biofilm matrix
components, many of which have yet to be studied in A. baumannii.
As the biofilm continues to develop and mature, the process is
believed to be linked to a ubiquitous intracellular messenger signaling
molecule cyclic-di-GMP, a cyclic-di-nucleotide recently identified in
A. baumannii regulating biofilm formation and surface associated
motility (Ahmad et al., 2020). Cyclic-di-GMP and cyclic-di-AMP have
been identified in other bacteria to be an allosteric activator of exopo­
lysaccharide biosynthesis which directly contributes to virulence and
biofilm expression of Vibrio cholerae and Pseudomonas aeruginosa, mak­
ing its disruption a viable option in therapeutic development (Pestrak
and Wozniak, 2020; Young et al., 2020). Furthermore, a key phenom­
enon that helps sustain the biofilm is quorum sensing (QS). Quorum
sensing is the mode of communication shared among the bacteria to
maintain the biofilm and population density via signal molecules called
auto inducers. Changes in expression and concentration in these auto
inducers as the biofilm matures through the increase in bacterial pop­
ulation results in signal transduction cascade that informs the bacteria
on how to best adapt (Saipriya et al., 2019). One well documented
means by which this is achieved is through the expression of N-acyl-­
homoserine lactones (AHL), which is an auto inducer signal molecule.
The QS occurs when AbaI, a sensor protein, functions as a synthase to
produce AHL signal molecule that bind with AbaR receptor to produce a
signal cascade. CsuA/BABCDE chaperone-usher system expression was
also upregulated when bacterial cells were cultured with non-native
AHLs, increasing biofilm density (Luo et al., 2015).
Some previously mentioned contributors in the development and
maturation of biofilm includes: blaPER-1 gene where positive expression
resulted in an increase in biofilm formation (Lee et al., 2008); Biofilm
associated protein (BAP) found to be widely present in many
A. baumannii strains (Goh et al., 2013) and biofilm like proteins (BLP)
whose gene disruption lead to impaired biofilm formation (De Gregario
et al., 2015). Overall, many virulence factors play a multi-concerted role
of both adhesion and biofilm formation, with any disruption of the
former leading to the absence of biofilm formation and thus an
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attenuated virulence profile. Therefore, although there still exists gap in
our knowledge of the biogenesis of A. baumannii biofilm formation, the
bulk of the work on biofilms has shown that it is an integral part of the
virulence and persistence strategy employed by the pathogen. As such, it
follows reasonably that the biofilm has been targeted for the develop­
ment of novel therapeutic strategies.
4. Host-pathogen interaction studies
Due to the urgent need to understand the pathogenesis and virulence
of the bacteria especially in understanding its interaction with host
during infection, studies employing host-pathogen interaction models
have been employed. It ranges from in vitro to in vivo models providing
the necessary means to evaluate novel treatments, characterize host
immune response and identify novel bacterial virulence factors. Animal
models in particular have been the mainstay in studying A. baumannii
pathogenesis especially due to the wide array of clinical manifestations
perpetrated by the pathogen. Two major challenges exist when
employing the use of animal models, primarily the ability to properly
and accurately mimic human disease and the bacteria strains used when
conducting these interaction studies.
4.1. In-vivo models
4.1.1. Mammalian models
The primary choice for mammalian in vivo studies has been to use
murine models with some studies employing other mammals such as
rabbits and guinea pigs although this is rather rare (Pachón-Ibáñez et al.,
2010). These studies were initially carried out to evaluate the antibiotic
efficacy during Acinetobacter infection but has since shifted to investi­
gate bacterial pathogenesis, host response & immunity along with new
alternative therapeutic strategies (Bentancor et al., 2012; Cerqueira
et al., 2014; Jolly-Guillou et al., 1997; Sánchez-Encinales et al., 2017;
Skerniškytė et al., 2019). Although, these studies could never realisti­
cally reproduce human disease infection process, they have nonetheless
provided a wealth of data to glean in our understanding of bacterial
pathogenesis, providing a clearer picture from whence to approach
effective infection control. In addition, the wide range and availability of
murine models coupled with cost and diverse use case solidifies it as a
staple infection model of choice. As A. baumannii can cause quite a broad
range of infections, it is imperative that a suitable selection of murine
strains be conducted to best acquire the closest resemblance to infections
in humans. Some more common inbred and outbred strains which have
been successfully used include A/J, BALB/c, C57BL/6 and Swiss
Webster mice. Whichever strain chosen can affect the outcome of the
infection studies, where similar ATCC® bacteria strains when used re­
sults in differences to mice survival, tissue load and clearance and im­
mune response (Álvarez-Fraga et al., 2018; Palmer et al., 2019).
4.1.2. Pneumonia model
As pneumonia represents the highest clinical manifestation of
A. baumannii infections, lung infection studies using murine models have
also steadily increased. The first study to have used this model was
performed in 1997 by Joly-Guillou and colleagues testing the efficacy of
imipenem against acute A. baumannii pneumonia. The goal of this model
is to introduce bacteria into the mouse in order to induce inflammatory
responses consistent with acute pneumonia. From there researchers are
also able to evaluate the virulence expression of the pathogen and have
made some interesting discoveries. So far, we identified two approaches
to achieve this immune response: intra-tracheal and intranasal in­
troductions of bacteria. The former involves using a blunt ended needle
to cannulate the trachea of an anesthetized mice inoculated with bac­
terial suspension, while the latter involves bacterial suspension is
directly applied to the nostrils as the mouse inhales breathing the sus­
pension in (Joly-Guillou et al., 1997; Palmer et al., 2019). Usually, these
models struggle to induce a proper infection in immunocompetent mice
H.J. Mea et al.
which leads to a difficulty in mimicking the actual disease as seen in
clinical settings. This is usually overcome by inducing neutropenia or
using neutropenic mice (Harris et al., 2013). However, some studies
have proposed that this step could be omitted if a sufficiently virulent
strain of bacteria is used, giving us greater insight into the pathogenesis
of the bacteria (Harris et al., 2013).
Studies have also shown the usefulness of pneumonia models in
highlighting the host-response to A. baumannii infections. This was
achieved by using transgenic mice with well-defined genetic changes
which allows for more specific characterization of host response on a
molecular level. One such example was the knockout of CD14 and Toll-
like receptors 2 and 4 resulting in a difficulty in bacterial clearance,
which highlights the role played by these receptors effective immune
response (Knapp et al., 2006). Another study using gp91phox− /− and
NOS− /− mice shows the essential role of NADPH phagocyte oxidase and
NOS2 has in limiting bacterial growth and replication (Qiu et al., 2008).
While using transgenic mice have shown great potential, as seen in these
examples, it has yet to be widely adopted in studying A. baumannii
pneumonia model. In addition, pneumonia model has also been used to
study the effectiveness of vaccines which saw immunization of immu­
nocompetent mice prior to pneumonia infection leading to a drop in
bacterial load post infection when compared to the non-immunized
group (Bentancor et al., 2012).
4.1.3. Sepsis models
Bacteremia represents the second most common clinical manifesta­
tion of nosocomial A. baumannii infections mainly triggered by host toll
-like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS).
In severe cases, this could be a result of bacterial dissemination into the
bloodstream from an initial pneumonic infection. This process has been
successfully demonstrated using murine pneumonia models where a
depletion of alveolar macrophages and neutrophils resulted in increased
morbidity and mortality with extrapulmonary bacterial dissemination
consistent with sepsis (de Breij et al., 2012 & Lee et al., 2020) To date,
sepsis models also employed the use of similar murine strains as used in
pneumonia models which generally involved the use of two main ap­
proaches. The first and most widely adopted model is the murine peri­
toneal sepsis model which saw extensive used in other pathogens such as
K. pneumoniae and later adopted in A. baumannii antibiotic and subse­
quently virulence studies (Ko et al., 2004; Smani et al., 2013; Tsai et al.,
2011). This model has been used extensively to establish minimum le­
thal doses (MLDs) part of characterizing the many clinical strains iso­
lated for pathogenesis and virulence studies which when compared to
ATCC® reference strains help to highlight the evolution undertaken by
the pathogen through the decades. A rarer approach involves the use of
inducing systemic infection via introducing the pathogen retro-orbitally
which has the added advantage of reducing complications and distress
from the animal though a highly skilled operator is required (Yardeni
et al., 2011). This approached has been successfully used to study the
role of TLR9 signaling in response to A. baumannii infection in both
pneumonic and systemic infection (Noto et al., 2015)
4.1.4. Non-mammalian models
Interestingly, although mammalian models still provide the best
means to study host pathogen interaction, interest in non-mammalian
models has recently grown as they offer several advantages over their
mammalian counterparts. Firstly, non-mammalian models are not
bound by ethical restrictions. Next, compared to mammalian models
which require costly holding facilities, non-mammalian models are
relatively cheaper. These factors allow researchers to employ large
quantities of organisms in any given study, facilitating high-throughput
screening for bacterial virulence factors, toxins and general bacterial
virulence. Such studies can utilize not only large number of host but a
diverse range of bacterial strains which would expedite the character­
ization of clinical strains as they emerge. Some of the models used thus
far include Caenorhabditis elegans (nematode), Dictyostelium discoideum
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(amoeba), Galleria mellonella (greater wax moth) and Danio rerio (zebra
fish).
Caenorhabditis elegans is a soil inhabitant nematode that is micro­
scopic in size with a length of approximately 1 mm when fully matured.
It has a short generation time of approximately 3 days and is able to
reproduce up to 400 progenies from a single adult as it is a hermaph­
rodite. As a result of its well characterized genome, anatomical
simplicity and transparent body, C. elegans is an appealing host for
infection studies as seen in studying bacterial pathogenesis of Salmonella
enterica, Listeria monocytogenes, Burkholderia pseudomallei and Legionella
pneumophila (Anju et al., 2020; Brassinga and Sifri, 2019). Though
simpler in nature, the innate immune system of the nematode has sur­
prising similarities with that of humans. Seen primarily in its conserve
immune regulators that facilitate host’ identification of pathogens at the
onset of infection. This allows for anti-infective agents screening which,
if found to be protective to the nematode, is useful as a potential com­
pound for further animal trials and subsequent human trials
(Skerniškytė et al., 2019). The first describe use of C. elegans to study A
baumannii was by Smith et al. (2004) which investigated the effects of
ethanol as a signaling molecule for various Acinetobacter spp. That study
found that ethanol positively triggered A. baumannii virulence which
negatively affected worm proliferation and brood size. Since then,
C. elegans have also been adopted to study eukaryote and prokaryote
relationships and anti-virulence characteristics of various compounds
(Khadke et al., 2019; Raorane et al., 2019). Interestingly, one study
modified Smith’s work and demonstrated the successful use of C. elegans
in A. baumannii pathogenesis study. Concluding that fertility assay was
more accurate than killing assay and validated by intraperitoneal in­
jection murine sepsis model (Vallejo et al., 2015) The only disadvantage
is the incubation temperature of C. elegans is relatively low at 25 ◦ C
compared to the usual growth temperature of A baumannii at 37 ◦ C. This
could affect its virulence and survivability translating into a possible
discrepancy.
Used alongside C. elegans, Dictyostelium discoideum is a unicellular
amoeba that has also been proposed mainly for its ability to feed on
bacteria by phagocytosis, an ability which lends itself to being used as a
model for human macrophage-bacterial interactions (Anju et al., 2020).
One of the major advantages is its ability to also grow at 37 ◦ C, allowing
for greater relevance to actual bacterial pathogenesis in human host.
One study aimed at identifying mutant A. baumannii strains, employed
the coculturing of amoeba with bacteria. Since bacteria grows faster
than the amoeba, it was able to form colonies first before consumption
by amoeba which forms visible plaques on the bacterial lawn. Bacteria
with plaques were successfully identified as mutant A. baumannii which
lack certain virulence factors allowing for rapid screening of transposon
mutant libraries for future pathogenesis studies (Smith et al., 2007). This
provides an interesting opportunity for rapid screening of large pools of
clinical isolates collected across countries around world for virulence
characterization.
Galleria mellonella is the caterpillar of the greater wax moth and has
been utilized in host-entomopathogenic microbe interaction studies
prior to its utilization as a model for human pathogens. Its low cost,
convenience and ethically acceptable ranking has made it rather
favorable for use as a model, in addition to its ability to grow well at 37
◦
C. It has been used extensively in several other pathogenic bacteria,
both gram positive and negative species with substantial utility in
A. baumannii studies as well (Anju et al., 2020; Peleg et al., 2009). Its
complex and unique immune system shares some similarities with the
mammalian immune system, specifically the presence of phagocytic
cells, antimicrobial peptides, encapsulation and clotting factors and
complement like proteins (Tsai et al., 2016). Its introduction as a host for
A. baumannii was demonstrated by Peleg (2009) and colleagues where
they demonstrated the direct correlation of host death to bacterial load
and prolonged survival when antibiotics was administered. Since then,
many more studies have incorporated this model in studies involving
antimicrobial efficacy, bacterial virulence expression and pathogenicity
H.J. Mea et al.
(Gaddy et al., 2012; Richmond et al., 2016; Sánchez-Encinales et al.,
2017; Skiebe et al., 2012).
Danio rerio, more commonly known as the zebrafish is a more recent
host-pathogen model with limited uses in A. baumannii infection studies.
Favored for its quick breeding and development with innate immune
cells appearing 25 h post fertilization. Studies into zebrafish have pri­
marily been focused on high throughput screening of drugs meant for
cancer, inflammatory diseases and infectious diseases due to some
orthologous genes shared with mammals (van der Sar et al., 2004).
Many studies have successfully used transgenic zebrafish with fluores­
cent tags attached to various immune cells allowing for greater in-depth
visualization of the immune response during infection (Tobin et al.,
2012; Zhang et al., 2019)). Thus far, one study which utilized Zebrafish
showed that A. baumannii mutant lacking ΔgacS led to neutrophils
migrating to the site of infection but failed to migrate away, suggesting
that there exists a bacterial driven metabolic by-product that was
responsible as a neutrophil chemotaxis. Bhuiyan et al. (2016) was able
to study the migration pattern using fluorophore-marked bacteria and
selective fluorescent immune cells to track their movements and in­
teractions within the host at real-time. This provides a whole new
avenue of study that can shed greater light on both host immune
response and bacterial immune evasion simultaneously. It should be
noted that studies employing these models cannot stand alone and
mammalian models will remain the mainstay approach to studying
A. baumannii virulence and pathogenesis. It can, however, serve to
provide rapid bacterial characterization of various clinical strains and
evaluation of novel antimicrobial efficacy.
4.2. In-vitro model
In addition to in vivo models, many studies aimed at elucidating
bacterial virulence and pathogenesis have also utilized in vitro models to
get a preliminary glimpse into the complex host-pathogen interactions.
Being simpler and more cost-effective, these studies have provided the
basis on which many in vivo studies may take place with complementing
data to substantiate any findings. Since pathogens first contact with a
given host upon successful entry is with the epithelial mucous mem­
brane cells, it has become the first choice in most in vitro studies. This is
true for a broad range of diseases and surfaces on the human body and
evaluating this interaction provides vital information in terms of path­
ogen virulence properties, immunomodulation and antimicrobial ac­
tivities in the given context as the infection progresses (Pedersen et al.,
2018).
Most of the in vitro studies involved use epithelial cells of a given part
of the body co-infected with a bacterial strain specific to the site of
infection. This is first to first assess the adherence and cytotoxicity
properties of the pathogen. Comparison is usually made between
reference ATCC® and clinically isolated contemporary strains which
provides important phenotypic information that can be further explored
through comparative genomic sequence analysis. A. baumannii adher­
ence and biofilm forming characteristics were identified in similar
fashion which led to future studies employing similar in vitro models in
order to assess the virulence of clinically isolated strains (Tomaras et al.,
2003). In vitro models also provide useful initial host immunological
response to the presence of pathogens as co-infection and bacterial
adherence to the host triggers an inflammatory response aimed
recruiting immune cells necessary for bacterial clearance. Though most
of the immune response to a foreign pathogen follows a general pattern,
some unique bacterial surface proteins are capable of elucidating a
varied response that give clues to the molecular mechanism behind
bacterial pathogenesis which can vary depending on the site of infection
(March et al., 2010; Vijayakumar et al., 2016). This variation can be
explained by the need of the bacteria to adapt to a given environment,
expressing virulence specifically catered to its needs for survival as can
be seen in biofilm and motility variations observed in A. baumannii
isolated from sputum or the blood (Vijayakumar et al., 2016).
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Recently, infection studies involving in-vitro models have seen the
use of a host cell line co-cultured with immune cells. It has been
established using murine models that immune cells are critical during
the early stages of infection, producing pro-inflammatory cytokines such
as TNF-α, IL-6, MCP-1 and MIP-2 in the lungs which supports the
clearance of A. baumannii infection (van Faassen et al., 2007; de Breij
et al., 2012). In-vitro studies utilizing just immune cells such as those
carried out by Qiu et al. (2012), help shed light on the important role
played by these immune cells which are part of the innate immune
system’s first response to infection with phagocytosis observed within
10 min of co-incubation. Interestingly, a later study incorporated both
macrophage and neutrophil co-culture with either A. baumannii or
A. pittii clinical and reference strains show that neutrophils are more
crucial in in controlling infection with it being more effective at killing
bacteria and essential for the recruitment and activation of macrophages
(Lázaro-Díez et al., 2017).The same research group further expanded on
this by elucidating the neutrophil’s response to A. baumannii infection
through gene expression and cytokine production assays uncovering the
upregulation and production of key pro-inflammatory mediators crucial
for effective infection control (Lázaro-Díez et al., 2020). These advances
demonstrate the growing knowledge and understanding of the not only
the pathogen but also host response which is a step in the right direction
and is critical for future translation to novel therapeutic strategies. Ul­
timately, cell lines provide important information not immediately
accessible from in vivo or clinical observations by limiting the scope to
molecular mechanisms expressed by the host and pathogen to establish a
successful infection. Providing useful data for understanding the infec­
tion process starting from the lungs extending to bacteremia, which to
the best of our knowledge remains an untapped avenue for research.
5. Conclusion future perspective
This review has shown the considerable advancement made in un­
derstanding the pathogenesis and virulence characteristics harbored by
A. baumannii in the initial stages of infection, but also comments on
some gaps that still exist. Much of the advances stem from the adoption
of many notable in vivo and in vitro models that allowed for an in-depth
study and characterization of various clinical strains isolated. This
coupled with a large catalogue of over 2000 genome sequence publicly
available, positions researchers to be more readily able to do large
comparative genomic studies in mapping the bacteria’s core genome
and compare the variations that exist around the world in terms of
virulence and pathogenicity. Additionally, categorization studies to
identify which clonal lineages the many strains belong to could serve to
expand our understanding of the spread of the pathogen along with its
evolution in acquiring multi-drug resistance. With evidence of genome
plasticity arising in this pathogen, the clock is ticking as we encounter an
increasing number of pan-drug resistant A. baumannii strains where
current existing antimicrobial agents are no longer capable at treating its
infections (Traglia et al., 2018). Thankfully, the current employment of
ATCC® 17978™ and ®ATCC 19606™ as reference standards serve well
as controls in many comparative studies. However, they do highlight the
need for consistency in identifying and characterizing clinically relevant
strains using well-established combined in vivo and in vitro models and
procedures highlighted in this review. With such a coordinated effort,
the direct comparison between studies would no doubt pave a promising
future as new innovative techniques continue to develop and optimize
allowing for a greater understanding of the pathogen, spearheading
development of more effective novel therapeutic strategies.
Author contributions
Hing Jian Mea carried out the literature search, planning and writing
of the manuscript draft. Eng Hwa Wong and Phelim Voon Chen Yong
edited and contributed to the structure and designed. All authors have
reviewed and approved the final manuscript draft before submission.
H.J. Mea et al. Microbiological Research 247 (2021) 126722

Funding Choi, C., Lee, E., Lee, Y., Park, T., Kim, H., Hyun, S., et al., 2005. Outer membrane
protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces
apoptosis of epithelial cells. Cell. Microbiol. 7 (8), 1127–1138.
This study was financially supported by the Taylor’s University Choi, C., Hyun, S., Lee, J., Lee, J., Lee, Y., Kim, S., et al., 2007. Acinetobacter baumannii
Research Grant – Scheme Project No: TRGS/MFS/2017/SBS/003; outer membrane protein A targets the nucleus and induces cytotoxicity. Cell.
TRGS/MFS/2/2014/SOM/005. The funders had no role in the design, Microbiol. 10 (2), 309–319. https://doi.org/10.1111/j.1462-5822.2007.01041.x.
Choi, C., Lee, J., Lee, Y., Park, T., Lee, J., 2008. Acinetobacter baumannii invades epithelial
collection, and analysis of the data, manuscript draft preparation, or cells and outer membrane protein A mediates interactions with epithelial cells. BMC
decision to publish. Microbiol. 8 (1), 216.
Choi, A., Slamti, L., Avci, F., Pier, G., Maira-Litrán, T., 2009. The pgaABCD Locus of
Acinetobacter baumannii Encodes the Production of Poly-β-1-6-N-
Declaration of Competing Interest Acetylglucosamine, Which Is Critical for Biofilm Formation. J. Bacteriol. 191 (19),
5953–5963.
Clemmer, K., Bonomo, R., Rather, P., 2011. Genetic analysis of surface motility in
The authors report no declarations of interest. Acinetobacter baumannii. Microbiology 157 (9), 2534–2544.
Corral, J., Pérez-Varela, M., Barbé, J., Aranda, J., 2020a. Direct interaction between
Acknowledgements RecA and a CheW-like protein is required for surface-associated motility, chemotaxis
and the full virulence of Acinetobacter baumannii strain ATCC 17978. Virulence 11
(1), 315–326.
This review was supported by Taylor’s University Research Grant – Corral, J., Sebastià, P., Coll, N., Barbé, J., Aranda, J., Valls, M., 2020b. Twitching and
Scheme Project No: TRGS/MFS/2017/SBS/003; TRGS/MFS/2/2014/ swimming motility play a role in Ralstonia solanacearum pathogenicity. Msphere 5
(2).
SOM/005. Dahdouh, E., Suarez Rodriguez, M., Daoud, Z., 2018. Comparison of virulence
determinants among Acinetobacter baumannii Clinical isolates obtained from Spain
References and Lebanon. J. Infect. Dev. 12 (02.1), 15S.
de Breij, A., Gaddy, J., van der Meer, J., Koning, R., Koster, A., van den Broek, P., et al.,
2009. CsuA/BABCDE-dependent pili are not involved in the adherence of
Ahmad, I., Karah, N., Nadeem, A., Wai, S., Uhlin, B., 2019. Analysis of colony phase
Acinetobacter baumannii ATCC19606T to human airway epithelial cells and their
variation switch in Acinetobacter baumannii clinical isolates. PLoS One 14 (1) p.
inflammatory response. Res. Microbiol. 160 (3), 213–218.
e0210082.
de Breij, A., Dijkshoorn, L., Lagendijk, E., van der Meer, J., Koster, A., Bloemberg, G.,
Ahmad, I., Nygren, E., Khalid, F., Myint, S., Uhlin, B., 2020. A Cyclic-di-GMP signalling
et al., 2010. Do biofilm formation and interactions with human cells explain the
network regulates biofilm formation and surface associated motility of Acinetobacter
clinical success of Acinetobacter baumannii? PLoS One 5 (5) p. e10732.
baumannii 17978. Sci. Rep. 10 (1).
de Breij, A., Eveillard, M., Dijkshoorn, L., van den Broek, P., Nibbering, P., Joly-
Álvarez-Fraga, L., Vázquez-Ucha, J., Martínez-Guitián, M., Vallejo, J., Bou, G.,
Guillou, M., 2012. Differences in Acinetobacter baumannii strains and host innate
Beceiro, A., Poza, M., 2018. Pneumonia infection in mice reveals the involvement of
immune response determine morbidity and mortality in experimental pneumonia.
the feoA gene in the pathogenesis of Acinetobacter baumannii. Virulence 9 (1),
PLoS One 7 (2) p. e30673.
496–509.
De Gregorio, E., Del Franco, M., Martinucci, M., Roscetto, E., Zarrilli, R., Di Nocera, P.,
Anju, V., Siddhardha, B., Dyavaiah, M., 2020. Animal models to understand
2015. Biofilm-associated proteins: news from Acinetobacter. BMC Genomics 16 (1).
Host–Pathogen interactions. Model Organisms For Microbial Pathogenesis. Biofilm
Dijkshoorn, L., Nemec, A., Seifert, H., 2007. An increasing threat in hospitals: multidrug-
Formation And Antimicrobial Drug Discovery, pp. 393–411.
resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5 (12), 939–951.
Aranda, J., Bardina, C., Beceiro, A., Rumbo, S., Cabral, M., Barbe, J., Bou, G., 2011.
Dorsey, C., Tomaras, A., Actis, L., 2002. Genetic and phenotypic analysis of
Acinetobacter baumannii RecA protein in repair of dNA damage, antimicrobial
Acinetobacter baumannii insertion derivatives generated with a transposome
resistance, general stress response, and virulence. J. Bacteriol. 193 (15), 3740–3747.
system. Appl. Environ. Microbiol. 68 (12), 6353–6360.
Bardbari, A., Arabestani, M., Karami, M., Keramat, F., Alikhani, M., Bagheri, K., 2017.
Dufour, D., Leung, V., Lévesque, C., 2010. Bacterial biofilm: structure, function, and
Correlation between ability of biofilm formation with their responsible genes and
antimicrobial resistance. Endod. Topics 22 (1), 2–16.
MDR patterns in clinical and environmental Acinetobacter baumannii isolates.
Eijkelkamp, B., Hassan, K., Paulsen, I., Brown, M., 2011a. Investigation of the human
Microb. Pathog. 108, 122–128.
pathogen Acinetobacter baumannii under iron limiting conditions. BMC Genomics
Barker, J., Maxted, H., 1975. Observations on the growth and movement of acinetobacter
12 (1).
on semi-solid media. J. Med. Microbiol. 8 (3), 443–446.
Eijkelkamp, B., Stroeher, U., Hassan, K., Papadimitrious, M., Paulsen, I., Brown, M.,
Bartholomew, T., Kidd, T., Sá Pessoa, J., Conde Álvarez, R., Bengoechea, J., 2019. 2-
2011b. Adherence and motility characteristics of clinical Acinetobacter baumannii
Hydroxylation of Acinetobacter baumannii lipid a contributes to virulence. Infect.
isolates. FEMS Microbiol. Lett. 323 (1), 44–51.
Immun. 87 (4).
Eijkelkamp, B., Stroeher, U., Hassan, K., Paulsen, I., Brown, M., 2014. Comparative
Beceiro, A., Llobet, E., Aranda, J., Bengoechea, J., Doumith, M., Hornsey, M., et al.,
analysis of surface-exposed virulence factors of Acinetobacter baumannii. BMC
2011. Phosphoethanolamine modification of lipid A in colistin-resistant variants of
Genomics 15 (1), 1020.
Acinetobacter baumannii mediated by the pmrAB two-component regulatory system.
Falagas, M., Rafailidis, P., 2007. Attributable mortality of Acinetobacter baumannii: no
Antimicrob. Agents Chemother. 55 (7), 3370–3379.
longer a controversial issue. Crit. Care 11 (3), 134.
Bentancor, L., Camacho-Peiro, A., Bozkurt-Guzel, C., Pier, G., Maira-Litran, T., 2012.
Fiester, S., Arivett, B., Schmidt, R., Beckett, A., Ticak, T., Carrier, M., et al., 2016. Iron-
Identification of Ata, a multifunctional trimeric autotransporter of Acinetobacter
regulated phospholipase C activity contributes to the cytolytic activity and virulence
baumannii. J. Bacteriol. 194 (15), 3950–3960.
of Acinetobacter baumannii. PLoS One 11 (11) p. e0167068.
Bhuiyan, M., Ellett, F., Murray, G., Kostoulias, X., Cerqueira, G., Schulze, K., et al., 2016.
Gaddy, J., Arivett, B., McConnell, M., López-Rojas, R., Pachón, J., Actis, L., 2012. Role of
Acinetobacter baumannii phenylacetic acid metabolism influences infection
acinetobactin-mediated Iron acquisition functions in the interaction of Acinetobacter
outcome through a direct effect on neutrophil chemotaxis. Proc. Natl. Acad. Sci. 113
baumannii strain ATCC 19606Twith human lung epithelial cells, galleria mellonella
(34), 9599–9604.
caterpillars, and mice. Infect. Immun. 80 (3), 1015–1024.
Boucher, H., Talbot, G., Bradley, J., Edwards, J., Gilbert, D., Rice, L., et al., 2009. Bad
Gebhardt, M., Shuman, H., 2017. GigA and GigB are master regulators of antibiotic
bugs, No drugs: No ESKAPE! An update from the infectious diseases society of
resistance, stress responses, and virulence in Acinetobacter baumannii. J. Bacteriol.
america. Clin. Infect. Dis. 48 (1), 1–12.
199 (10).
Brassinga, A., Sifri, C., 2019. The Caenorhabditis elegans model of Legionella infection.
Giammanco, A., Calà, C., Fasciana, T., Dowzicky, M., 2017. Global assessment of the
Methods Mol. Biol. 371–397.
activity of tigecycline against multidrug-resistant gram-negative pathogens between
Brisou, J., Prevot, A., 1954. Studies on bacterial taxonomy. X. The revision of species
2004 and 2014 as part of the tigecycline evaluation and surveillance trial. Msphere 2
under Acromobacter group. Ann. Inst. Pasteur (Paris) 86 (6), 722–728.
(1).
Brossard, K., Campagnari, A., 2011. The Acinetobacter baumannii biofilm-associated
Goh, H., Beatson, S., Totsika, M., Moriel, D., Phan, M., Szubert, J., et al., 2013. Molecular
protein plays a role in adherence to human epithelial cells. Infect. Immun. 80 (1),
analysis of the Acinetobacter baumannii biofilm-associated protein. Appl. Environ.
228–233.
Microbiol. 79 (21), 6535–6543.
Centre of Disease Control and Prevention, 2019. Antibiotic Threats in the United States.
Green, E., Juttukonda, L., Skaar, E., 2019. The manganese-responsive transcriptional
Cerqueira, G., Kostoulias, X., Khoo, C., Aibinu, I., Qu, Y., Traven, A., Peleg, A., 2014.
regulator MumR protects Acinetobacter baumannii from oxidative stress. Infect.
A global virulence regulator in Acinetobacter baumannii and its control of the
Immun. 88 (3).
phenylacetic acid catabolic pathway. J. Infect. Dis. 210 (1), 46–55.
Greene, C., Vadlamudi, G., Newton, D., Foxman, B., Xi, C., 2016a. The influence of
Chapartegui-González, I., Lázaro-Díez, M., Bravo, Z., Navas, J., Icardo, J., Ramos-
biofilm formation and multidrug resistance on environmental survival of clinical and
Vivas, J., 2018. Acinetobacter baumannii maintains its virulence after long-time
environmental isolates of Acinetobacter baumannii. Am. J. Infect. Control 44 (5),
starvation. PLoS One 13 (8) p. e0201961.
e65–e71.
Chen, R., Lv, R., Wang, M., Du, Z., Tan, Y., Cui, Y., 2017. A1S_2811, a CheA/Y-like
Greene, C., Wu, J., Rickard, A., Xi, C., 2016b. Evaluation of the ability of Acinetobacter
hybrid two-component regulator from Acinetobacter baumannii ATCC17978, is
baumannii to form biofilms on six different biomedical relevant surfaces. Lett. Appl.
involved in surface motility and biofilm formation in this bacterium.
Microbiol. 63 (4), 233–239.
MicrobiologyOpen 6 (5), e00510. https://doi.org/10.1002/mbo3.510. In press.
Groisman, E., 2016. Feedback control of two-component regulatory systems. Annu. Rev.
Chin, C., Gregg, K., Napier, B., Ernst, R., Weiss, D., 2015. A PmrB-Regulated deacetylase
Microbiol. 70 (1), 103–124.
required for lipid a modification and polymyxin resistance in Acinetobacter
baumannii. Antimicrob. Agents Chemother. 59 (12), 7911–7914.

10
H.J. Mea et al.
Harding, C., Kinsella, R., Palmer, L., Skaar, E., Feldman, M., 2016. Medically relevant
Acinetobacter species require a type II secretion system and specific membrane-
associated chaperones for the export of multiple substrates and full virulence. PLoS
Pathog. 12 (1) p. e1005391.
Harris, G., Kuo Lee, R., Lam, C., Kanzaki, G., Patel, G., Xu, H., Chen, W., 2013. A mouse
model of Acinetobacter baumannii-associated pneumonia using a clinically isolated
hypervirulent strain. Antimicrob. Agents Chemother. 57 (8), 3601–3613.
Hospenthal, M., Costa, T., Waksman, G., 2017. A comprehensive guide to pilus
biogenesis in Gram-negative bacteria. Nat. Rev. Microbiol. 15 (6), 365–379.
Hoyle, B., Costerton, J., 1991. Bacterial Resistance to Antibiotics: the Role of Biofilms.
Progress In Drug Research / Fortschritte Der Arzneimittelforschung / Progrès Des
Recherches Pharmaceutiques, pp. 91–105.
Jarrell, K., McBride, M., 2008. The surprisingly diverse ways that prokaryotes move. Nat.
Rev. Microbiol. 6 (6), 466–476.
Jin, J., Kwon, S., Moon, D., Gurung, G., Lee, J., Kim, S., et al., 2011. Acinetobacter
baumannii secretes cytotoxic outer membrane protein A via outer membrane
vesicles. PLoS One 6 (2), e17027. https://doi.org/10.1371/journal.pone.0017027.
In press.
Joly-Guillou, M., Wolff, M., Pocidalo, J., Walker, F., Carbon, C., 1997. Use of a new
mouse model of Acinetobacter baumannii pneumonia to evaluate the postantibiotic
effect of imipenem. Antimicrob. Agents Chemother. 41 (2), 345–351.
Josenhans, C., Suerbaum, S., 2002. The role of motility as a virulence factor in bacteria.
Int. J. Med. Microbiol. 291 (8), 605–614.
Jun, S., Lee, J., Kim, B., Kim, S., Park, T., Lee, J., Lee, Y., 2013. Acinetobacter baumannii
outer membrane vesicles elicit a potent innate immune response via membrane
proteins. PLoS One 8 (8) p. e71751.
Khadke, S., Lee, J., Woo, J., Lee, J., 2019. Inhibitory effects of Honokiol and magnolol on
biofilm formation by Acinetobacter baumannii. Biotechnol. Bioprocess Eng. 24 (2),
359–365.
khelissa, S., Jama, C., Abdallah, M., Boukherroub, R., Faille, C., Chihib, N., 2017. Effect
of incubation duration, growth temperature, and abiotic surface type on cell surface
properties, adhesion and pathogenicity of biofilm-detached Staphylococcus aureus
cells. AMB Express 7 (1).
Kim, S., Oh, M., Jun, S., Jeon, H., Kim, S., Kim, K., et al., 2016. Outer membrane Protein
A plays a role in pathogenesis of Acinetobacter nosocomialis. Virulence 7 (4),
413–426.
Kim, S., Kim, M., Kim, S., Son, J., Kim, S., Lee, Y., et al., 2019. The sensor kinase BfmS
controls production of outer membrane vesicles in Acinetobacter baumannii. BMC
Microbiol. 19 (1).
Knapp, S., Wieland, C., Florquin, S., Pantophlet, R., Dijkshoorn, L., Tshimbalanga, N.,
et al., 2006. Differential roles of CD14 and toll-like receptors 4and 2 in
MurineAcinetobacterPneumonia. Am. J. Respir. Crit. Care Med. 173 (1), 122–129.
Ko, W., Lee, H., Chiang, S., Yan, J., Wu, J., Lu, C., Chuang, Y., 2004. In vitro and in vivo
activity of meropenem and sulbactam against a multidrug-resistant Acinetobacter
baumannii strain. J. Antimicrob. Chemother. 53 (2), 393–395.
Koenigs, A., Zipfel, P., Kraiczy, P., 2015. Translation elongation factor tuf of
Acinetobacter baumannii is a plasminogen-binding protein. PLoS One 10 (7) p.
e0134418.
Krasowska, A., Sigler, K., 2014. How microorganisms use hydrophobicity and what does
this mean for human needs? Front. Cell. Infect. Microbiol. 4.
Kröger, C., Kary, S., Schauer, K., Cameron, A., 2017. Genetic regulation of virulence and
antibiotic resistance in Acinetobacter baumannii. Genes 8 (1), 12.
Kwon, H., Kim, S., Oh, M., Shin, M., Lee, J., 2019. Distinct role of outer membrane
protein A in the intrinsic resistance of Acinetobacter baumannii and Acinetobacter
nosocomialis. Infect. Genet. Evol. 67, 33–37.
Lázaro-Díez, M., Navascués-Lejarza, T., Remuzgo-Martínez, S., Navas, J., Icardo, J.,
Acosta, F., et al., 2016. Acinetobacter baumannii and A. Pittii clinical isolates lack
adherence and cytotoxicity to lung epithelial cells in vitro. Microbes Infect. 18 (9),
559–564.
Lázaro-Díez, M., Chapartegui-González, I., Redondo-Salvo, S., Leigh, C., Merino, D.,
Segundo, D., et al., 2017. Human neutrophils phagocytose and kill Acinetobacter
baumannii and A. Pittii. Sci. Rep. 7 (1).
Lázaro-Díez, M., Chapartegui-González, I., Suberbiola, B., Ocejo-Vinyals, J., López-
Hoyos, M., Ramos-Vivas, J., 2020. Gene expression profiling in human neutrophils
after infection with Acinetobacter baumannii in vitro. PLoS One 15 (11) p.
e0242674.
Lee, J., Oh, J., Kim, K., Jeong, Y., Park, J., CHO, J., 2001. Apoptotic cell death induced by
Acinetobacter baumannii in epithelial cells through caspase-3 activation note. APMIS
109 (10), 679–684.
Lee, J., Koerten, H., van den Broek, P., Beekhuizen, H., Wolterbeek, R., van den
Barselaar, M., et al., 2006. Adherence of Acinetobacter baumannii strains to human
bronchial epithelial cells. Res. Microbiol. 157 (4), 360–366.
Lee, H., Koh, Y., Kim, J., Lee, J., Lee, Y., Seol, S., et al., 2008. Capacity of multidrug-
resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to
epithelial cell surfaces. Clin. Microbiol. Infect. 14 (1), 49–54.
Lees-Miller, R., Iwashkiw, J., Scott, N., Seper, A., Vinogradov, E., Schild, S., Feldman, M.,
2013. A common pathway forO-linked protein-glycosylation and synthesis of capsule
inAcinetobacter baumannii. Mol. Microbiol. 89 (5), 816–830.
Lin, M., Lin, Y., Yeh, H., Lan, C., 2014. Role of the BaeSR two-component system in the
regulation of Acinetobacter baumannii adeAB genes and its correlation with
tigecycline susceptibility. BMC Microbiol. 14 (1), 119.
Liou, M., Soo, P., Ling, S., Kuo, H., Tang, C., Chang, K., 2014. The sensor kinase BfmS
mediates virulence in Acinetobacter baumannii. J. Microbiol. Immunol. Infect. 47
(4), 275–281.
11
Microbiological Research 247 (2021) 126722
Loehfelm, T., Luke, N., Campagnari, A., 2008. Identification and characterization of an
Acinetobacter baumannii biofilm-associated protein. J. Bacteriol. 190 (3),
1036–1044.
Lonergan, Z., Nairn, B., Wang, J., Hsu, Y., Hesse, L., Beavers, W., et al., 2019. An
Acinetobacter baumannii, zinc-regulated peptidase maintains cell wall integrity
during immune-mediated nutrient sequestration. Cell Rep. 26 (8), 2009–2018 e6.
Luo, L., Wu, L., Xiao, Y., Zhao, D., Chen, Z., Kang, M., et al., 2015. Enhancing pili
assembly and biofilm formation in Acinetobacter baumannii ATCC19606 using non-
native acyl-homoserine lactones. BMC Microbiol. 15 (1).
Mattick, J., 2002. Type IV pili and twitching motility. Annu. Rev. Microbiol. 56 (1),
289–314.
May, H., Yu, J., Shrihari, S., Seshu, J., Klose, K., Cap, A., et al., 2019. Thioredoxin
modulates cell surface hydrophobicity in Acinetobacter baumannii. Front. Microbiol.
10.
McQueary, C., Kirkup, B., Si, Y., Barlow, M., Actis, L., Craft, D., Zurawski, D., 2012.
Extracellular stress and lipopolysaccharide modulate Acinetobacter baumannii
surface-associated motility. J. Microbiol. 50 (3), 434–443.
Montaña, S., Vilacoba, E., Traglia, G., Almuzara, M., Pennini, M., Fernández, A., et al.,
2015. Genetic variability of AdeRS two-component system associated with
tigecycline resistance in XDR-Acinetobacter baumannii isolates. Curr. Microbiol. 71
(1), 76–82.
Moriel, D., Beatson, S., Wurpel, D., Lipman, J., Nimmo, G., Paterson, D., Schembri, M.,
2013. Identification of novel vaccine candidates against multidrug-resistant
Acinetobacter baumannii. PLoS One 8 (10) p. e77631.
Nait Chabane, Y., Marti, S., Rihouey, C., Alexandre, S., Hardouin, J., Lesouhaitier, O.,
et al., 2014. Characterisation of pellicles formed by Acinetobacter baumannii at the
air-liquid interface. PLoS One 9 (10) p. e111660.
Noto, M., Boyd, K., Burns, W., Varga, M., Peek, R., Skaar, E., 2015. Toll-like receptor 9
contributes to defense against Acinetobacter baumannii infection. Infect. Immun. 83
(10), 4134–4141.
Pachón-Ibáñez, M., Docobo-Pérez, F., López-Rojas, R., Dominguez-Herrera,
́ J., Jiménez-
Mejias, M., Garcia-Curiel,
́ A., et al., 2010. Efficacy of rifampin and its combinations
with imipenem, sulbactam, and colistin in experimental models of infection caused
by Imipenem-Resistant Acinetobacter baumannii. Antimicrob. Agents Chemother. 54
(3), 1165–1172.
Pakharukova, N., Tuittila, M., Paavilainen, S., Zavialov, A., 2017. Methylation,
crystallization and SAD phasing of the Csu pilus CsuC–CsuE chaperone–adhesin
subunit pre-assembly complex from Acinetobacter baumannii. Acta Crystallogr. F
Struct. Biol. Commun. 73 (8), 450–454.
Pakharukova, N., Tuittila, M., Paavilainen, S., Malmi, H., Parilova, O., Teneberg, S.,
et al., 2018. Structural basis for Acinetobacter baumannii biofilm formation. Proc.
Natl. Acad. Sci. 115 (21), 5558–5563.
Palmer, L., Green, E., Sheldon, J., Skaar, E., 2019. Assessing Acinetobacter baumannii
virulence and persistence in a murine model of lung infection. Methods Mol. Biol.
289–305.
Park, Y., Lee, J., Ko, K., 2015. Transcriptomic analysis of colistin-susceptible and colistin-
resistant isolates identifies genes associated with colistin resistance in Acinetobacter
baumannii. Clin. Microbiol. Infect. 21 (8), 765.e1–765.e7.
Pedersen, R., Grønnemose, R., Stærk, K., Asferg, C., Andersen, T., Kolmos, H., et al.,
2018. A method for quantification of epithelium colonization capacity by pathogenic
Bacteria. Front. Cell. Infect. Microbiol. 8.
Peleg, A., Seifert, H., Paterson, D., 2008. Acinetobacter baumannii: emergence of a
successful pathogen. Clin. Microbiol. Rev. 21 (3), 538–582.
Peleg, A., Jara, S., Monga, D., Eliopoulos, G., Moellering, R., Mylonakis, E., 2009.
Galleria mellonella as a model system to study Acinetobacter baumannii
pathogenesis and therapeutics. Antimicrob. Agents Chemother. 53 (6), 2605–2609.
Pendleton, J., Gorman, S., Gilmore, B., 2013. Clinical relevance of the ESKAPE
pathogens. Expert Rev. Anti. Ther. 11 (3), 297–308.
Pérez, A., Merino, M., Rumbo-Feal, S., Álvarez-Fraga, L., Vallejo, J., Beceiro, A., et al.,
2016. The FhaB/FhaC two-partner secretion system is involved in adhesion
ofAcinetobacter baumanniiAbH12O-A2 strain. Virulence 8 (6), 959–974.
Pérez-Varela, M., Corral, J., Vallejo, J., Rumbo-Feal, S., Bou, G., Aranda, J., Barbé, J.,
2017. Mutations in the β-Subunit of the RNA polymerase impair the surface-
associated motility and virulence of Acinetobacter baumannii. Infect. Immun. 85 (8).
Pestrak, M., Wozniak, D., 2020. Regulation of cyclic di-GMP signaling in Pseudomonas
aeruginosa. Microbial Cyclic Di-Nucleotide Signal. 471–486.
Petersen, K., Riddle, M., Danko, J., Blazes, D., Hayden, R., Tasker, S., Dunne, J., 2007.
Trauma-related infections in battlefield casualties from Iraq. Ann. Surg. 245 (5),
803–811.
Petrova, O., Sauer, K., 2012. Sticky situations: key components that control bacterial
surface attachment. J. Bacteriol. 194 (10), 2413–2425.
Piepenbrink, K., Lillehoj, E., Harding, C., Labonte, J., Zuo, X., Rapp, C., et al., 2016.
Structural diversity in the type IV pili of multidrug-resistant Acinetobacter. J. Biol.
Chem. 291 (44), 22924–22935.
Pour, N., Dusane, D., Dhakephalkar, P., Zamin, F., Zinjarde, S., Chopade, B., 2011.
Biofilm formation byAcinetobacter baumanniistrains isolated from urinary tract
infection and urinary catheters. FEMS Immunol. Med. Microbiol. 62 (3), 328–338.
Qiu, H., KuoLee, R., Harris, G., Chen, W., 2008. Role of NADPH phagocyte oxidase in
host defense against acute respiratory Acinetobacter baumannii infection in mice.
Infect. Immun. 77 (3), 1015–1021.
Qiu, H., KuoLee, R., Harris, G., Van Rooijen, N., Patel, G., Chen, W., 2012. Role of
macrophages in early host resistance to respiratory Acinetobacter baumannii
infection. PLoS One 7 (6) p. e40019.
Raorane, C., Lee, J., Kim, Y., Rajasekharan, S., García-Contreras, R., Lee, J., 2019.
Antibiofilm and Antivirulence Efficacies of flavonoids and curcumin against
Acinetobacter baumannii. Front. Microbiol. 10.
H.J. Mea et al.
Rello, J., Kalwaje Eshwara, V., Lagunes, L., Alves, J., Wunderink, R., Conway-Morris, A.,
et al., 2018. A global priority list of the TOp TEn resistant Microorganisms (TOTEM)
study at intensive care: a prioritization exercise based on multi-criteria decision
analysis. Eur. J. Clin. Microbiol. Infect. Dis. 38 (2), 319–323.
Repizo, G., Gagné, S., Foucault-Grunenwald, M., Borges, V., Charpentier, X.,
Limansky, A., et al., 2015. Differential role of the T6SS in Acinetobacter baumannii
virulence. PLoS One 10 (9) p. e0138265.
Richmond, G., Evans, L., Anderson, M., Wand, M., Bonney, L., Ivens, A., et al., 2016. The
Acinetobacter baumannii two-component system AdeRS regulates genes required for
multidrug efflux, biofilm formation, and virulence in a strain-specific manner. Mbio
7 (2).
Ronish, L., Lillehoj, E., Fields, J., Sundberg, E., Piepenbrink, K., 2018. The structure of
PilA from Acinetobacter baumannii AB5075 suggests a mechanism for functional
specialization in Acinetobacter type IV pili. J. Biol. Chem. 294 (1), 218–230.
Rosenberg, E., DeLong, E., Lory, S., Stackebrandt, E., Thompson, F., 2013. The
Prokaryotes: Human Microbioogy, 4th edn. Springer, Berlin, Heidelberg,
pp. 107–123.
Rumbo, C., Tomás, M., Fernández Moreira, E., Soares, N., Carvajal, M., Santillana, E.,
et al., 2014. The Acinetobacter baumannii Omp33-36 porin is a virulence factor that
induces apoptosis and modulates autophagy in human cells. Infect. Immun. 82 (11),
4666–4680.
Rumbo-Feal, S., Gómez, M., Gayoso, C., Álvarez-Fraga, L., Cabral, M., Aransay, A., et al.,
2013. Whole transcriptome analysis of Acinetobacter baumannii assessed by RNA-
Sequencing reveals different mRNA expression profiles in biofilm compared to
planktonic cells. PLoS One 8 (8) p. e72968.
Russo, T., Luke, N., Beanan, J., Olson, R., Sauberan, S., MacDonald, U., et al., 2010. The
K1 capsular polysaccharide of Acinetobacter baumannii strain 307-0294 is a major
virulence factor. Infect. Immun. 78 (9), 3993–4000.
Sahu, P., Iyer, P., Oak, A., Pardesi, K., Chopade, B., 2012. Characterization of eDNA from
the Clinical StrainAcinetobacter baumanniiAIIMS 7 and its Role in Biofilm
Formation. Sci. World J. 2012, 1–10.
Sánchez-Encinales, V., Álvarez-Marín, R., Pachón-Ibáñez, M., Fernández-Cuenca, F.,
Pascual, A., Garnacho-Montero, J., et al., 2017. Overproduction of outer membrane
protein A (OmpA) by Acinetobacter baumannii is a risk factor for nosocomial
pneumonia, bacteremia and mortality increase. J. Infect. Dis. p. jix010.
Sato, Y., Unno, Y., Kawakami, S., Ubagai, T., Ono, Y., 2017. Virulence characteristics of
Acinetobacter baumannii clinical isolates vary with the expression levels of omps",
in. J. Med. Microbiol. 66 (2), 203–212.
Silverman, J., Brunet, Y., Cascales, E., Mougous, J., 2012. Structure and regulation of the
type VI secretion system. Annu. Rev. Microbiol. 66 (1), 453–472.
Skerniškytė, J., Krasauskas, R., Péchoux, C., Kulakauskas, S., Armalytė, J.,
Sužiedėlienė, E., 2019. Surface-related features and virulence among Acinetobacter
baumannii clinical isolates belonging to international clones I and II. Front.
Microbiol. 9.
Skiebe, E., de Berardinis, V., Morczinek, P., Kerrinnes, T., Faber, F., Lepka, D., et al.,
2012. Surface-associated motility, a common trait of clinical isolates of
Acinetobacter baumannii, depends on 1,3-diaminopropane. Int. J. Med. Microbiol.
302 (3), 117–128.
Smani, Y., Docobo-Pérez, F., López-Rojas, R., Domínguez-Herrera, J., Ibáñez-
Martínez, J., Pachón, J., 2012a. Platelet-activating Factor Receptor Initiates Contact
of Acinetobacter baumanniiExpressing Phosphorylcholine with Host Cells. J. Biol.
Chem. 287 (32), 26901–26910.
Smani, Y., McConnell, M., Pachón, J., 2012b. Role of fibronectin in the adhesion of
Acinetobacter baumannii to host cells. PLoS One 7 (4) p. e33073.
Smani, Y., Dominguez-Herrera, J., Pachon, J., 2013. Association of the outer membrane
protein Omp33 with fitness and virulence of Acinetobacter baumannii. J. Infect. Dis.
208 (10), 1561–1570.
Smith, M., Des Etages, S., Snyder, M., 2004. Microbial synergy via an ethanol-triggered
pathway. Mol. Cell. Biol. 24 (9), 3874–3884.
Smith, M., Gianoulis, T., Pukatzki, S., Mekalanos, J., Ornston, L., Gerstein, M.,
Snyder, M., 2007. New insights into Acinetobacter baumannii pathogenesis revealed
by high-density pyrosequencing and transposon mutagenesis. Genes Dev. 21 (5),
601–614.
Sun, J., Chiang, Y., Shang, H., Perng, C., Yang, Y., Chiueh, T., 2017. Phenotype
microarray analysis of the AdeRS two-component system in Acinetobacter
baumannii. Eur. J. Clin. Microbiol. Infect. Dis. 36 (12), 2343–2353.
12
Microbiological Research 247 (2021) 126722
Tacconelli, E., Carrara, E., Savoldi, A., Harbarth, S., Mendelson, M., Monnet, D., et al.,
2018. Discovery, research, and development of new antibiotics: the WHO priority
list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18 (3),
318–327.
Talà, L., Fineberg, A., Kukura, P., Persat, A., 2019. Pseudomonas aeruginosa orchestrates
twitching motility by sequential control of type IV pili movements. Nat. Microbiol. 4
(5), 774–780.
Thibau, A., Dichter, A., Vaca, D., Linke, D., Goldman, A., Kempf, V., 2019.
Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine
targets. Med. Microbiol. Immunol. 209 (3), 243–263.
Tipton, K., Rather, P., 2017. An ompR-envZ two-component system ortholog regulates
phase variation, osmotic tolerance, motility, and virulence in Acinetobacter
baumannii strain AB5075. J. Bacteriol. 199 (3).
Tobin, D., May, R., Wheeler, R., 2012. Zebrafish: a see-through host and a fluorescent
toolbox to probe host–pathogen interaction. PLoS Pathog. 8 (1) p. e1002349.
Tomaras, A., Dorsey, C., Edelmann, R., Actis, L., 2003. Attachment to and biofilm
formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel
chaperone-usher pili assembly system. Microbiology 149 (12), 3473–3484.
Traglia, G., Chiem, K., Quinn, B., Fernandez, J., Montaña, S., Almuzara, M., et al., 2018.
Genome sequence analysis of an extensively drug-resistant Acinetobacter baumannii
indigo-pigmented strain depicts evidence of increase genome plasticity. Sci. Rep. 8
(1).
Tsai, Y., Fung, C., Lin, J., Chen, J., Chang, F., Chen, T., Siu, L., 2011. Klebsiella
pneumoniaeOuter membrane porins OmpK35 and OmpK36 play roles in both
antimicrobial resistance and virulence. Antimicrob. Agents Chemother. 55 (4),
1485–1493.
Tsai, C., Loh, J., Proft, T., 2016. Galleria mellonella infection models for the study of
bacterial diseases and for antimicrobial drug testing. Virulence 7 (3), 214–229.
Vallejo, J., Beceiro, A., Rumbo-Feal, S., Rodríguez-Palero, M., Russo, T., Bou, G., 2015.
Optimisation of the Caenorhabditis elegans model for studying the pathogenesis of
opportunistic Acinetobacter baumannii. Int. J. Antimicrob. Agents.
van der Sar, A., Appelmelk, B., Vandenbroucke-Grauls, C., Bitter, W., 2004. A star with
stripes: zebrafish as an infection model. Trends Microbiol. 12 (10), 451–457.
van Faassen, H., KuoLee, R., Harris, G., Zhao, X., Conlan, J., Chen, W., 2007. Neutrophils
play an important role in host resistance to respiratory infection with Acinetobacter
baumannii in mice. Infect. Immun. 75 (12), 5597–5608.
Vij, R., Crawford, C., Casadevall, A., 2019. Variation in cell surface hydrophobicity
among Cryptococcus neoformans strains influences interactions with amoeba.
Msphere.
Vijayakumar, S., Rajenderan, S., Laishram, S., Anandan, S., Balaji, V., Biswas, I., 2016.
Biofilm formation and motility depend on the nature of the Acinetobacter baumannii
clinical isolates. Front. Public Health 4.
Weidensdorfer, M., Chae, J., Makobe, C., Stahl, J., Averhoff, B., Müller, V., et al., 2016.
Analysis of endothelial adherence of Bartonella henselae and Acinetobacter
baumannii using a dynamic HumanEx vivo infection model. Infect. Immun. 84 (3),
711–722.
Whitchurch, C., Tolker-Nielsen, T., Rages, P., Mattick, J., 2002. Extracellular DNA
required for bacterial biofilm formation. Science 295 (5559), pp. 1487-1487.
Wilharm, G., Piesker, J., Laue, M., Skiebe, E., 2013. DNA uptake by the nosocomial
pathogen Acinetobacter baumannii occurs during movement along wet surfaces.
J. Bacteriol. 195 (18), 4146–4153.
Wood, C., Ohneck, E., Edelmann, R., Actis, L., 2018. A light-regulated type I pilus
contributes toAcinetobacter baumanniiBiofilm, motility, and virulence functions.
Infect. Immun. 86 (9).
Yang, L., Barken, K., Skindersoe, M., Christensen, A., Givskov, M., Tolker-Nielsen, T.,
2007. Effects of iron on DNA release and biofilm development by Pseudomonas
aeruginosa. Microbiology 153 (5), 1318–1328.
Yang, C., Su, P., Moi, S., Chuang, L., 2019. Biofilm formation in Acinetobacter
baumannii: genotype-phenotype correlation. Molecules 24 (10), 1849.
Young, E., Bonds, G., Karatan, E., 2020. Signals modulating cyclic di-GMP pathways in
Vibrio cholerae. Microb. Cyclic Di-Nucl. Signal. 357–378.
Zhang, X., de Boer, L., Stockhammer, O., Grijpma, D., Spaink, H., Zaat, S., 2019.
A zebrafish embryo model for in vivo visualization and intravital analysis of
biomaterial-associated Staphylococcus aureus infection. J. Vis. Exp. (143) p. e58523.
Zhang, L., Liang, W., Xu, S., Mei, J., Di, Y., Lan, H., et al., 2020. CarO promotes adhesion
and colonization of Acinetobacter baumannii through inhibiting NF-кB pathways.
Int. J. Clin. Exp. Med. 12 (3), 2518–2524.