Infection, Genetics and Evolution 112 (2023) 105459

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Infection, Genetics and Evolution

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Review

Efflux pumps and microbial biofilm formation

Mahdyeh Neghabi Hajiagha a, Hossein Samadi Kafil b, *
a
Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
b
Drug Applied Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

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

Keywords: Biofilm-related infections are resistant forms of pathogens that are regarded as a medical problem, particularly
Biofilm due to the spread of multiple drug resistance. One of the factors associated with biofilm drug resistance is the
Efflux pumps presence of various types of efflux pumps in bacteria. Efflux pumps also play a role in biofilm formation by
Drug resistance
influencing Physical-chemical interactions, mobility, gene regulation, quorum sensing (QS), extracellular poly­
Infection
meric substances (EPS), and toxic compound extrusion. According to the findings of studies based on efflux pump
Pathogenesis
expression analysis, their role in the anatomical position within the biofilm will differ depending on the biofilm
formation stage, encoding gene expression level, the type and concentration of substrate. In some cases, the
function of the efflux pumps can overlap with each other, so it seems necessary to accurate identify the efflux
pumps of biofilm-forming bacteria along with their function in this process. Such studies will help to choose
treatment strategy, at least in combination with antibiotics. Furthermore, if the goal of treatment is an efflux
pump manipulation, we should not limit it to inhibition.

1. Introduction
Antibiotic resistance has emerged in bacteria, raising the problem of
recalcitrant infections (Aslam et al., 2018; Ventola, 2015). Multidrug
resistant (MDR) bacteria is a public health crisis that negatively affects
the clinical outcomes, particularly in immunocompromised patients or
those hospitalized in intensive care units (Bialvaei et al., 2016; Vivas
et al., 2019) (Chen et al., 2020; Gasperini et al., 2021; Wang et al., 2017;
Wang et al., 2019b). The rapid increase in the development of genetic
basis of antibiotic resistance has been regarded as the primary cause of
this phenomenon. However, bacteria are smart organized microorgan­
isms that they can also enter to a physiologically dormant state and
display phenotypic tolerance to antibiotic treatment. They can manage
complicated situations in different environments and correlation with
other competitions. So, in order to survive and colonise effectively, they
employ a variety of strategies. (Maisonneuve and Gerdes, 2014). Several
important molecular mechanisms whereby bacteria use them to tolerate
and persistence in stress conditions include: RpoS (Schellhorn, 2020)
and the general stress response (Hecker and Völker, 2001), the SOS
response (Zgur Bertok and Podlesek, 2020), tolerance to reactive oxygen
species (ROS) (Johnson and Hug, 2019), energy metabolism/cell
respiration(oxygen oxidoreduction by Cytochrome bd) (Borisov et al.,
2020) and Tau metabolism (Javaux et al., 2007), the stringent response
((p)ppGpp signaling) (Irving and Corrigan, 2018; Pacios et al., 2020),
toxin-antitoxin (TA) system (Ronneau and Helaine, 2019), drug efflux
pumps (Kumar et al., 2020), the quorum sensing (QS) system as bacterial
communication process (Mukherjee and Bassler, 2019).
Biofilms are constituted of several different microbial species (pol­
ymicrobial) (Kafil and Mobarez, 2015a; Wolcott et al., 2013) that are
contained in a three-dimensional extracellular matrix made up of
extracellular polymeric substances (EPS) (Karygianni et al., 2020) and
work synergistically. This matrix, which is known as “safe haven”, serves
as a biological barrier against antimicrobial agents and immune com­
ponents, while still having a secure ecological niche for microorganisms
to survive (Kumar et al., 2019). Indeed, biofilm communities shelter
resistant and persister cells that can tolerate antibiotics or antimicrobial
agents and regrow when the antibiotic is withdrawn (Conlon et al.,
2015; Fisher et al., 2017). Microbial biofilm formation is now recog­
nized as a significant virulence factor in several localized primary in­
fections (Kafil and Mobarez, 2015b; Kafil et al., 2013; Mohamad et al.,
2023). Nevertheless, evidence that tolerant and persister bacteria play a
significant role in biofilm-related bacterial relapse and infection recal­
citrance has been increasing in recent years (Yan and Bassler, 2019).
Efflux pumps are proteins found in the cytoplasmic membrane of all
cells, including bacteria and eukaryotes (Van Bambeke et al., 2003a;
Van Bambeke et al., 2003b). These are responsible for secreting toxins or

* Corresponding author at: Drug Applied Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
E-mail address: kafilhs@tbzmed.ac.ir (H.S. Kafil).

https://doi.org/10.1016/j.meegid.2023.105459
Received 13 April 2023; Received in revised form 25 May 2023; Accepted 27 May 2023
Available online 2 June 2023
1567-1348/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.N. Hajiagha and H.S. Kafil
antibiotics formed by the cell, as well as the efflux of toxic compounds
found in the bacterial context, such as antibiotics (Marquez, 2005).
Despite the fact that bacterial efflux pumps play a significant role in
bacterial resistance, their involvement in bacterial infection dissemi­
nation via biofilm formation cannot be overlooked (Ebbensgaard et al.,
2020; Webber and Piddock, 2003). Therefore, in current study, we will
investigate the development of biofilm, the effect of bacterial efflux
pumps on it, and the associated antimicrobial resistance in order to
develop a novel treatment strategy, such as multi-targeted or combi­
national therapies.
2. Biofilm
William J. Costerton coined the word "biofilm" to describe the
aggregate existence of bacteria adhering to surfaces in 1978 (Costerton
et al., 1978). Biofilm analysis has developed into a common subject of
research within microbiology since the beginning of that accomplish­
ment (Koo and Yamada, 2016). At that time, the number of articles
published on biofilm and public health topics has increased from 1459 in
1992 to 7303 in 2004–2016 (Donlan, 2016). In this regard biofilm
known as architectural colony made by microorganisms such as pro­
karyotes (bacteria) and eukaryotes such as fungi and microalgae (mostly
revealed) (Ramage et al., 2012; Yuan et al., 2021) that have adapted
their metabolism for the respective environment (Yuan et al., 2021). The
key cause of cell attachment to surfaces and aggregation is the extra­
cellular matrix, which is the consequence of the cells’ natural production
of extracellular polymeric substance (EPS). The multiple gradients that
occur within biofilm matrix create a micro-niche (heterogeneous mi­
croenvironments) and provide a framework of cell-to-cell interactions
(Flemming et al., 2016).
2.1. Biofilm-related infections:
Biofilms are now recognized as widely distributed, existing in
aquatic and agricultural water systems, and also a wide range of eco­
systems and medical instruments with public health implications
(Donlan, 2002; Kordmahaleh and Shalke, 2013). Microbial biofilm for­
mation, which was first defined as a plural behavior of microbial com­
munities, is now considered as a crucial virulence factor extensively in
various localized chronic and even systemic infections including those of
the auditory, cardiovascular, gastrointestinal, integumentary, repro­
ductive, respiratory, and urinary systems (Larsen and Fiehn, 2017;
Miller et al., 2021; Vestby et al., 2020). On the other aspect, beneficial
outcomes of biofilm formation on the body have been mentioned, such
as biofilm formation by commensal bacteria such as Staphylococcus
epidermidis, which stimulates the immune system and thereby inhibits
pathogenic bacteria colonization (Motta et al., 2021; Nguyen et al.,
2017). According to the Centers for Disease Control (CDC) and the Na­
tional Institutes of Health (NIH), biofilm formation is associated to 65
percent and 85 percent of all infections, particularly chronic infections
(Potera, 1999).
During a period of infection tranquilization, four possible causes for
the recurrence of biofilm - associated infections by bacteria are inter­
preted: 1. Biofilm may lead to the adoption of slow growth states such as
dormancy and acts as a reservoir of cells in a VBNC state in starvation
zones of the biofilm. 2. The features of the biofilm matrix may contribute
to tolerance by trapping or inactivating antimicrobials. 3. Antimicrobial
resistance may even arise in biofilm communities due to horizontal gene
transfer, which allows resistance genes to spread among cells. 4. In
certain conditions, the ’biogeography of infection’ can change the
steady progress of infection by altering spatial organization and evalu­
ating population communication in polymicrobial biofilm infections
(Flemming et al., 2016; Stacy et al., 2016). In some sorts of circum­
stances, bacterial infection is definitely linked to biofilm formation
(Table 1).
2
Infection, Genetics and Evolution 112 (2023) 105459
2.2. Biofilm formation
The development of anti-biofilm strategies is based on a thorough
comprehension of the biofilm formation process and biofilm-related
mechanical parameters. (Boudarel et al., 2018). Biofilm formation can
be induced by a multitude of endogenous and environmental criteria,
including bacterial cell density, nutritional status, and cellular stress,
which mostly varies throughout bacteria (Landini et al., 2010; O’Toole
et al., 2000). Naturally bacteria must approach a surface sufficiently
close (10–20 nm) to enhance their chances of adhering. They cling to a
surface either passively or actively, depending on whether they are
nonmotile bacteria, such as staphylococci or motile bacteria, such as
Pseudomonas aeruginosa (Joo and Otto, 2012). Despite the repellent
forces between the bacteria and the environmental surfaces, attractive
van der Waals and electrostatic interactions and the mechanical force of
flagella and fimbriae preparations on the other hand provides the link­
age (Palmer et al., 2007).
Biofilm development cycle proceeds in 3 main stages (Fig. 1): 1.
Establishment; consist of 2 phases involving 1.1. Reversible attachment,
also known as early adhesion in which bacteria use extracellular or­
ganelles and proteins, such as flagella, pili, fimbriae, curli fibres, and
outer membrane proteins, to sense and attach to surfaces. It is also
influenced by hydrophobic interactions and surface texture. The intra­
cellular levels of (c-diGMP) can affect to regulation of this process
(Hengge, 2009). 1.2. Irreversible attachment between bacteria and tis­
sue or inorganic texture and as well as cell-cell adhesion is enabled by
secretion of an extracellular polymeric substance (EPS) or slime that
consists of DNA (eDNA), proteins, lipids, and lipopolysaccharides,
siderophores between cells and surfaces (Flemming and Wingender,
2010; Passos da Silva et al., 2017). 2. The second phase is growth, which
has two stages: proliferation and maturation. 2.1. Proliferation that in
this process, adherent bacteria multiply and expand to form three-
dimensional microcolonies (10–100μ). In through the synthesis of EPS
and the polymerization of a viscoelastic matrix, this community is pre­
served from the extracellular environment. Quorum sensing (QS) or
chemical cell-cell communication as the complex regulatory process
relies on high-density plays a key role in modulating of production and
composition of EPS, and monitoring their extracellular environment.
2.2. Mature phase that bacteria replicate and clump together in EPS to
form a three-dimensional structure that protects cells from outside
pressures. This structure is mushroom or tower like; shape for example
in P. aeruginosa (Klausen et al., 2003). Additional biofilm constituents,
including proteins, DNA, and polysaccharides, will accumulate as the
biofilm develops (Koo and Yamada, 2016; Rabin et al., 2015). On the
other hand Intercellular communication in the biofilm allows bacteria to
acquire specific functions and the bacteria are replaced in different
biofilm layers based on their metabolic and respiratory characteristics
(Rabin et al., 2015). Intercellular degradation factors generate path­
ways, which provide essential nutrients to lower cells and properly
dispose of waste (O’Toole et al., 2000). 3. Dispersal as the last stage that
some mature biofilm cells begin to separate and spread into the liquid
phase in duration (McDougald et al., 2012). This stage is critical because
separated cells (planktonic) might possibly start a new cycle of biofilm
formation. Biofilms spread for a variety of reasons, including nutritional
deficiency, competitive pressures, population outgrowth, and so forth
(Davies, 2011; McDougald et al., 2012; Rabin et al., 2015).
Depending on the type of pathogen, different factors are involved in
each stage of biofilm development, some of which are mentioned in
Fig. 1 (Schulze et al., 2021). Kimberly K. Jefferson in 2004 reported a
collection of genes involved in biofilm development (the cause genes), as
well as genes that are regulated differently in a developed biofilm (the
effect genes) (Jefferson, 2004). Furthermore, several experiments have
shown that the genes transcribed by biofilm-associated microbes vary
from those transcribed by planktonic counterparts (Donlan, 2002).
The biofilm is positively and negatively regulated by several physi­
cochemical factors that will be discussed in the following (Kafil et al.,
 M.N. Hajiagha and H.S. Kafil
Table 1
Biofilm related infections and their most common cause’s bacteria.

Medical
devises Cosmetic
Dental Voice
related Cerebro Tympanostomy Intravenous Biliary Intrauterine Joint
infections

implants prostheses filler

Tubes Dentures Tracheostomy Breast implant
spinal shunts catheter tract stents devices prosthesis
Contact Lenses Speech valve Tubes
Vascular prosthesis and Penile implants Orthopaedic fixation
Nasolacrimal silastic sten prostheses Endotracheal tubes
grafts Urinary tract catheters devices
Ocular abiotic materials
Prosthetic heart valves
Glaucoma tubes
Heart assist devices
Punctual plugs
Cardiac pacemakers
Corned sutures
Scleral buckle
Implanted crystalline
Cochlear implant
Bone-anchored hearing aid
implants
Tissue Chronic otitis media Caries Chronic rhinosinusitis Endocarditis on native Peptic ulcer disease Chronic prostatitis Chronic osteomyelitis Acne
infections Keratitis Chronic Adenoiditis valve Cystitis Necrotising fasciitis
Diffuse lamellar keratitis periodontitis Tonsillitis Pyelonephritis Chronic wounds
3

Endophthalmitis pneumonia Cystic fibrosis Urinary stones Peritoneal dialysis

Sinusitis Adenoid disease pneumonia Bacterial vaginosis catheter
Cholesteatoma Chronic obstructive Burn-related infection
Otitis Media pulmonary disease
(COPD)
Diffuse panbronchiolitis
Bronchicetasia
Common P. aeruginosa MRSA Oral flora P. aeruginosa E. faecalis H. pylori E. coli S. aureus P. acnes
causes CoNS S. pneumonia, R. dentacariosa K. pneumonia Bacteroidetes K. pneumonia S. pneumonie S. pyogenes
bacteria MRSA P. aeruginosa C. albicans P. aeruginosa Enterobacteriaceae P. aeruginosa P. acne S. aureus
Enterococcus spp S. aureus C. tropicalis S. aureus Fusobacterium spp P. mirabilis Propionibacterium spp Klebsiella spp
S. aureus H. influenza R. dentacariosa S. epidermidis Pseudomonadaceae Corynebacterium Clostridium spp
Streptococcus spp P. aeruginosa S. salivarius Streptococcus spp. Bacteroides

Infection, Genetics and Evolution 112 (2023) 105459

S. aureus P. aeruginosa
S. epidermidis
S. pneumonia,
Acinetobacter spp
M. catarrhalis
H. influenza
K. pneumonie Entrobacter
spp
M.N. Hajiagha and H.S. Kafil Infection, Genetics and Evolution 112 (2023) 105459

Fig. 1. Schematic representation of the three major stages of biofilm formation, from adherence to dispersion. In early stages motile or non-motile bacteria adhere to
either biotic or abiotic surfaces through the adhesion factors. The second stage continues with proliferation of attached bacteria and production of multifunctional
biofilm matrix which surrounds the cells. Finally, in third stage cells get released from the mature and 3D structure to re-build a new community.

2016; Liu et al., 2020; Renner and Weibel, 2011; Schulze et al., 2021): 1.
Surface physical characteristics electrostatic interactions and surface
energy are examples of physical interactions that impact cell attach­
ment. Some of these interactions and interfacial forces are hydration
forces, hydrophobic interactions, and steric forces.2. Chemical charac­
teristics have an impact on cell adherence to surfaces and the biofilm
formation. Chemical communications between cells and other signalling
factors including divalent cationic ions (e.g., Ca2+, Mg2+), nutrients,
quorum sensing systems (QS), regulatory small RNAs (sRNAs), alterna­
tive sigma factors, two-component systems and second messengers, such
as c-di-GMP and cAMP-CRP influences the organization of communities.
3. Efflux pumps related to multidrug resistance
Efflux pumps are proteinaceous transporter machinery system that
localized within the microorganism’s cell membrane. They are usually
associated with multidrug resistance due to their ability in extruding of
the several types of antibiotics in either solitude or cooperative manner
in bacteria. Moreover, each of efflux pumps can transport a variety of
structurally diverse substances, such as drugs, toxins and metabolites, or
they might be specialized for a single substrate.
Up to now, six families of bacterial efflux pumps have been estab­
lished to extract internal or entered substances from the bacterial

Fig. 2. Schematic illustration of six families of bacterial transporters along with their energy sources and location in the cell membrane. The major facilitator su­
perfamily (MFS), ATP-binding cassette (ABC), small multidrug resistance (SMR), and major facilitator superfamily (MATE) families are found in both gram-positive
and gram-negative species, whereas the resistance nodulation-cell division (RND) superfamily is present only in gram-negative species. The structure of the Pro­
teobacterial antimicrobial compound efflux (PACE) family has not been determined well yet. OM, outer membrane; IM, inner membrane; S, substrate.

4
M.N. Hajiagha and H.S. Kafil
cytoplasm and transport it toward the external environment (Fig. 2):
ATP-binding cassette (ABC) supplies the required energy to operate by
direct hydrolysis of ATP. The multidrug and toxin extrusion (MATE)
family has been composed of the Na+/H+ drug antiport systems, and
the major facilitator superfamily (MFS), the small multidrug resistance
(SMR) family, the resistance-nodulation-cell division (RND) superfamily
and the proteobacterial antimicrobial compound efflux (PACE) family
which work by the proton motive force.
They are usually encoded by chromosomal genes, but they also can
be expressed by transmissible genetic elements in some bacteria. The
gene expression of efflux pumps is regulated by complex and multi­
functional systems mediated by local and global regulators. These reg­
ulators are commonly located as repressors upstream of the operon that
encodes these genes. Inhibition or mutation of any of the positive or
negative regulators, for example, might alter the expression pathway
and result in overexpression.
Generally, regulation systems classified in three different types:
regulation by two-component systems, regulation by transcription and
post-transcription factors and regulation of AcrB by a small protein.
These systems are species-specific and function as an interconnected
network that is followed by the expression of other pumps if one pumps
stops expressing. They are also connected with many other cellular
processes at the transcriptional and posttranscriptional level. Accord­
ingly, deletion of these transporters not only affects antibiotic resistance
but also decreases or disrupts the other functions like adhesion, colo­
nization and dissemination, metabolism, invasion, motility, cell–cell
communication and biofilm formation.
There is apparently no detailed and accurate experimental data on
how efflux pumps contribute to biofilm formation except in a few cases.
Blocking (deleting or mutation) the efflux pump expression genes
(Matsumura et al., 2011), as well as inhibiting the efflux pumps them­
selves with efflux pump inhibitors in one or two different strains (de
Aguiar Coletti et al., 2017; Kvist et al., 2008), provides information on
the role of efflux pumps. It usually results in defects or a decline in
biofilm formation. However, enhanced biofilm formation has been
observed in rare cases, which might be caused by harmful substances
being trapped inside the bacteria. Another group of studies compares the
efflux pump’s expressing genes in biofilm-producing cells and persistent
Fig. 3. Possible roles of efflux pumps in biofilm development process. This division of functions based on the stage of biofilm formation is not certain and they can
overlap with each other as well.
5
Infection, Genetics and Evolution 112 (2023) 105459
with cells unable to form biofilms or planktonic cells. In most cases,
either up-regulation or overexpression and sometimes downregulation
are observed. A number of articles have compared the expression of
genes in biofilm-producing and planktonic cells in the presence or
absence of antibiotics with each other. On the other hand, investigation
of biofilm producing ability either in sensitive or antibiotic resistant
strains have been conducted in a few studies.
Therefore, it can be said that the most important role of efflux pumps
in biofilm formation process is to create ideal conditions or at least help
to establish such conditions. Several functions through which these
pumps can perform their role have been proposed based on their fea­
tures (Fig. 3): 1. Enhancement of biofilm matrix construction via
extruding of the extracellular polymeric substances, QS, and quorum
quenching (QQ) molecules, 2. Alteration in the expression of genes
involved in biofilm formation indirectly, 3. Excretion of harmful sub­
stances such as secondary metabolites, drugs, toxins and possibly host
immune molecules, 4. Facilitation of cell adhesion and aggregation
directly, 5. Efflux of biofilm formation stimulating factors which have
important role in biofilm formation like the iron acquisition agents, 6.
Influence on osmotic regulation and electrical charge of the bacterial
surface and around the cell by transit of minerals and ions.
Accurate identification of each efflux pump family and discovery of
their special role in biofilm formation can help to specifically inhibit it
and subsequently prevent biofilm development. Since multidrug resis­
tance is recognized as the most important feature of drug efflux pumps,
thus we will discuss the relationship between this feature and biofilm
formation in some bacterial strains first. In most cases, upregulation of
pumps encoding genes and increased resistance in biofilm cells have
been reported, but in some cases, it has also been seen to be down­
regulated. Physiological heterogeneity within the biofilm probably leads
to such a pattern of gene expression (Babin et al., 2017; Sankaran et al.,
2019; Stewart and Franklin, 2008).
Indeed, the expression of these genes depends on several factors,
including the type and concentration of substrate, the stage of biofilm
formation, the anatomical location of the biofilm in which the bacteria
are located, and the environmental conditions in which the biofilm is
formed (Fig. 4). For example, there is an increased expression of specific
antibiotic resistance pumps in surface-attached communities and
M.N. Hajiagha and H.S. Kafil Infection, Genetics and Evolution 112 (2023) 105459

Fig. 4. Suggested states for function of the efflux pump in biofilm formation based on the type of pump and substrate in different stages. a| First a pump with a wide
range of substrates, the role of which likely varies depending on environmental conditions, anatomical location, and the substance present in each step. b| The second
option is associated with pumps that can transport a multi-functional molecule (regulator, signal molecule, part of the EPS compound or toxic in high concentration).
The pump’s expression level controls the required functional concentration of the compound in each phase, or likewise, variable concentrations of the compound at
each level influence the pump’s expression rate. c| The final model is pumps with entirely specific substrates, which are expressed individually at each stage and
function in a specialized manner.

planktonic bacteria but decreased or no change in the expression of the
same gene in biofilm cells in the middle of the biofilm. In the middle of
the biofilm, however, there is an increase in the expression of pumps
involved in the release of secondary metabolites to remove waste
products. In terms of formation stage, increasing the expression of
pumps involved in adhesion in the early stages of biofilm formation or
increasing the expression of pumps involved in extracellular polymer
formation can be recorded in the growth and maturation stages of
biofilm.
Observing each phenomenon in different stages overlaps with each
other not far from expectation, so examining each pump carefully to
select it for a hypothetical therapeutic purpose is highly recommended.
According to studies, it seems two RND superfamily and MFS of efflux
pumps play a greater role in the biofilm formation process, although
more studies in this regard seem necessary. Therefore, we will first
describe the RND family and two members of this family and their role
in biofilm specific antibiotic resistance.
3.1. RND transporters
This family consists of a tripartite combined apparatus (Alvarez-
Ortega et al., 2013; Du et al., 2018; Tseng et al., 1999), constitutes of an
outer membrane protein channel, a periplasmic membrane-fusion
(adaptor) protein and a trimer cytoplasm-bound membrane pump.
Various classes of these types of efflux pumps have particularly defined
in Gram negative bacteria. Acr pumps of Escherichia coli and Mex pumps
of P. aeruginosa are two well-characterized members of this superfamily.
They actively extrude substrates by using the proton motive force as
their energy source.
The RND superfamily comprises mainly seven types that are the
heavy metal efflux (HME), the hydrophobe/amphiphile efflux-1 (Gram-
negative bacteria), the nodulation factor exporter family (NFE), the
SecDF protein-secretion accessory protein family, the hydrophobe/
amphiphile efflux-2 family, the eukaryotic sterol homeostasis family,
and the hydrophobe/amphiphile efflux-3 family. The idea of the evo­
lution process of RND pumps due to selection pressure in response to the
presence of antibiotics has been limited as a result of functional in­
vestigations and phylogenetic assessments. Researchers claim that these
transporters and associated diverse range of compounds (substrate
polyspecificities) evolved from physiological activities.
So far different roles of these pumps have been reported in cell ho­
meostasis that the most prominent of which include engagement in
bacterial virulence, interactions between bacteria and plants, trafficking
of quorum sensing molecules (intercellular communication). As well as,
they contribute in detoxification by efflux of either indigenous meta­
bolic intermediates or exogenous toxic compounds such as the antibi­
otics, heavy metals and solvents as the growth inhibitors and extrude the
produced antimicrobial substances.
The broad polyspecificity is due to either structural character of
RND’s binding pockets and transition channels like dynamics of the
loops, a broad range of ligands followed by different binding areas due
to the large size of binding pocket, hydration, and activated ligands with
aromatic side chains. The significance of two well-known RND efflux
pumps in biofilm-specific antibiotic resistance will be discussed in the

6
M.N. Hajiagha and H.S. Kafil
following sections.
3.1.1. AcrAB-TolC
AcrAB-TolC in E.coli and other Enterobacteriaceae is one of the first
described RND efflux pumps that are associated with increased levels of
multidrug resistance (Bialek-Davenet et al., 2014; Thanassi et al., 1997).
The Acr pump may play a role in biofilm resistance to ciprofloxacin at
low concentrations (Maira-Litrán et al., 2000). However, at the depth of
the biofilm, which has both a most suppressed growth rate and a lower
concentration of antibiotics due to its lower diffusion (Gilbert and
McBain, 2001), upregulation of the expression of multidrug resistance
(mar) operons and, as a result, the expression of Acr efflux pumps is
evident (Gilbert and McBain, 2001; Weston et al., 2018). This suggests
that Acr’s primary function in these cells cannot be associated with
increased antibiotic resistance in biofilm. There are also other studies
that support this idea by dismissing the release of antimicrobials as the
first function of AcrAB pumps in bacteria (Bialek et al., 2010; Buckner
et al., 2016).
Türkel et al. (2018) also examined the expression of AcrA in cipro­
floxacin susceptible and resistant strains of ESBL-producing Klebsiella
pneumoniae by using quantitative real-time PCR. They also compared the
ability of the two groups to form biofilms. This group’s findings revealed
that ciprofloxacin susceptible strains had higher levels of AcrA expres­
sion than resistant ones. Sensitive strains, on the other hand, had a
higher propensity for forming biofilms. However, the expression of
efflux genes had no effect on biofilm growth and no difference in efflux
gene expression was observed among biofilm production. Based on the
results of their research, the group suggested that the bacterium tries to
maintain itself in ciprofloxacin-sensitive conditions with minimal en­
ergy consumption by using biofilm formation or expression of efflux
pumps (Türkel et al., 2018). According to another investigation, there
was no difference between MDR and non-MDR K. pneumoniae strains in
their capacity to form biofilms. It’s even been found that high resistance
profiles were associated with decreased biofilm formation in both Aci­
netobacter baumannii and Salmonella enterica (Fabrega et al., 2014; Perez,
2015). In addition, the expression of efflux pumps genes such as acrA,
emrB, oqxA, and qacEΔ1 in K. pneumoniae is increased in biofilm
development.
As a consequence of the results, authors concluded that there is no
association between the quantity and type of efflux pump genes and
biofilm formation capacity, ruling out the involvement of efflux pump
expression as the primary determinant of biofilm resistance. They also
observed that using of concentrations above 10 mg of carbonyl
cyanidem-chlorophenyl hydrazine (CCCP) as an inhibitor of efflux
pumps further inhibits biofilm (Tang et al., 2020). Conversely, when the
CCCP concentration was less than 10 mg, biofilm formation was
induced. There are possibly some feedback mechanisms that aid to in­
crease EPS production or expression of additional efflux pump gene to
compensate for the blocked efflux pumps.
3.1.2. TolC as a component of RND and MFS efflux pumps
In E. coli, some members of RND efflux pumps, along with other
families, form multidrug resistance transporters (MDTs) that are
responsible for the extracellular efflux of compounds and also connect
the cytoplasm to periplasm and outer membrane (Saier Jr, 2000). In
some of these transporters TolC as an outer membrane protein link to the
RND members AcrB, AcrD, AcrF and MFS (EmrB) via periplasmic linkers
(AcrA, AcrE and EmrA) (Piddock, 2006). MDTs are responsible for the
phenomenon of multidrug resistance (MDR) in planktonic cells of E. coli.
Nevertheless several studies showed they are associated with the sessile
communities and their antibmicrobial resistance pattern as well (Ito
et al., 2009; Masadeh et al., 2013).
In the presence of subinhibitory concentrations of various antibi­
otics, studies on biofilm formation in E. coli revealed an increase in
biofilm formation (Penesyan et al., 2020) and the induction of expres­
sion of several genes encoding efflux pumps (Ebbensgaard et al., 2020;
7
Infection, Genetics and Evolution 112 (2023) 105459
Yasufuku et al., 2011). This resistance isn’t entirely due to the efflux of
toxic compounds through these transporters. The increased expression
of MDT pumps can be attributed to the response to stressful conditions
following the presence of antibiotics (Ma et al., 1995; Zhu et al., 2020).
In this regard Baye et al., showed in the presence of tetracycline and
tobramycin antibiotics, AcrD and AcrE mutant strains had similar or
enhanced biofilm growth and viability (Bay et al., 2017). They also
exhibited higher antibiotic resistance than planktonic to tested antibi­
otics (Bay et al., 2017).
However, E. coli (ΔtolC, ΔacrA and ΔacrB) strains exhibited reduction
in biofilm growth and approximately 2-fold increased antimicrobial
susceptibility compared with wild type (Bay et al., 2017). Even in a
similar study, impaired biofilm growth was observed for acrD and emrE
mutants (Matsumura et al., 2011). As well as, antimicrobial RND pump
inhibitor (verapamil) and the antimicrobial photodynamic therapy
(aPDT) mediated by methylene blue (MB) were assessed in Ecoli biofilm
(de Aguiar Coletti et al., 2017). It was found that following the appli­
cation of 22 J/cm2, 312 μg/mL of VP, and 200 μg/mL of MB, 80% of
metabolism reduction and 3.4 log10 CFU/mL declined for E. coli. In
interpreting these conflicting results, we can point to the possible dif­
ferences in the conditions of the two experiments.
Likely, a nutritional conditions and growth stages of biofilm play a
more important role in its antibacterial resistance than efflux activity of
pumps, and b. MDTs can indirectly affect biofilm resistance profile
through their effect on biofilm formation, growth and survival by the
overlapping specificities (Tal and Schuldiner, 2009). Nevertheless,
several research have reported pumps that may be efficient in biofilm
specific resistance, and recognizing such pumps can be extremely ad­
vantageous in biofilm control. For example, PA1874–1877 in
P. aeruginosa has been identified in Pseudomonas biofilm cells, and
removing the encoding genes resulted in tobramycin, gentamicin, and
ciprofloxacin sensitivity in these cells (Zhang and Mah, 2008)
3.1.3. MexAB-OprM
MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM are
another well-known RND pumps that contribute in antibiotic resistance
in P. aeruginosa. The role of this efflux pump in a biofilm-specific anti­
biotic resistance has also been reported (Mah and O’Toole, 2001; Olsen,
2015). It has been observed that expression of MexAB-oprM and/or
MexCD-oprJ in the presence of azithromycin pre-inhibitory concentra­
tions is essential for biofilm formation (Gillis et al., 2005). No elevation
in resistance to other antibiotics was observed in azithromycin-resistant
biofilm variants (Gillis et al., 2005). The expression of these pumps in
some AZM-resistant isolates can be greatly influenced by the mutation of
a negative nfxB regulator or not (Mulet et al., 2009). Although, the
sequencing of nfxB from PAO1 biofilm variant (PAO1-BV) in this study
showed that overexpression of MexCD-oprJ can’t be caused by a mu­
tation in nfxB (Gillis et al., 2005). It is possible that regulation of RND
efflux pumps are very complex and other factors like brlR, PA0756–0757
and rapA besides nfgx act as regulators which their mutations can lead to
biofilm-specific resistance (Hall and Mah, 2017). For example the ndvB
gene encodes a glucosyltransferase involved in the synthesis of cyclic-b-
(1, 3)-glucans (Bhagwat et al., 1996). They bind to tobramycin and
restrict it from reaching the cellular target as a novel mechanism of
resistance (Mah et al., 2003).
It has been demonstrated that mutants resistant to AZM exhibit
resistance towards ciprofloxacin and cefepime as well (Macia et al.,
2006; Mulet et al., 2009). This could be a warning about the effect of
AZM maintenance therapy on increasing drug resistance in P. aeruginosa
isolates from chronic respiratory infections due to overproduction of the
MexCD-OprJ and biofilm formation. In a distinct study that focused on
the ability of bacterial biofilm formation, particularly in those with
multidrug resistance, upregulation wasn’t observed in the expression of
these two pump’s genes. Interestingly, some instances of down­
regulation were actually documented (De Kievit et al., 2001). However,
some other studies have attributed the resistance of biofilm cells,
M.N. Hajiagha and H.S. Kafil Infection, Genetics and Evolution 112 (2023) 105459

especially bacteria in the substratum region, to other types of antibi­
otics, including tetracycline, chloramphenicol, quinolones, β-lactams,
aztreonam, gentamicin, tetracycline and tobramycin and colistin, to the
MexAB-oprM pump (Soto, 2013). MexEF-oprN and MexCD-oprJ other
members of this family contribute in biofilm antibiotic resistance in
areas with low oxygen tension (Schaible et al., 2012; Van Acker and
Coenye, 2016). The MexEF-oprN coding genes exhibited increased
expression in hypoxic cells of the biofilm, whereas no such result was
observed for the mexCD-oprJ pump genes (Schaible et al., 2012).
4. Role of efflux pumps in biofilm formation
4.1. Role in early stage
The early stages of biofilm formation are some complex events that
began by sense some especial environmental signals in bacteria. These
environmental signals differ from organism to organism. The initial
attachment of bacteria includes of physical and chemical interactions
that contribute to cell-surface and cell-cell interactions. Bacterial efflux
pumps and their components can involve in either physical (direct or
indirect effect via influence on attachment structures like pili) or
chemical interactions (efflux of effective chemical substances). They
may have some preventing or promoting role in aggregation. Accord­
ingly, depending on their role, they may show up or down-regulation.
Some efflux pumps and their likely substrate and function in the early
stages has been gathered in Table 2.
4.1.1. Role in physical and chemical interaction
Attachment of bacterial cells to any types of surfaces is influenced by
surface charges and hydrophobicity (Dickson and Koohmaraie, 1989).
Due to negatively charge of the most bacterial cell surfaces, attachment
and biofilm formation in positively charged surfaces is more favorable
(Song et al., 2015). However, some bacteria, such as Pseudomonas, are
able to modify their surface charge to form long-term biofilm structure
on negatively charged surfaces (Rzhepishevska et al., 2013). Hydro­
phobicity has both negative and advantageous effects on bacterial bio­
film formation capacity on biotic and abiotic surfaces (Pandey et al.,
2022).
The CusABCF pump in E. coli and other Gram-negative bacteria is
responsible for protecting them against lethal concentrations of Cu+ and
Ag+ by leaving it outside the bacterium (Greene and Koronakis, 2021;
Yun et al., 2010). The presence of low amount of copper or silver ions
around the bacteria can be thought to cause the environment to progress
towards a positive charge. Previously investigations has mentioned that
subinhibitory concentrations of some metal can contribute in cell sur­
face adhesion and/or cell-to-cell aggregation process and changes in
biofilm extracellular polymeric substance (EPS) matrix (de Araújo and
de Oliveira, 2019). Therefore, when selecting these toxic metals as
antibacterial compounds, proper application dosage should be
considered.
The ability of bacteria to overcome the osmolarity pressure of the
environment promotes the adhesive strength (Liu et al., 2013; Van der
Waal et al., 2011). There are two general strategies of osmoadaptation
using by bacteria to control hyper-osmotic conditions: a) the salt in
cytoplasm and b) the organic osmolyte such as proline, glycine betaine
and choline (Csonka, 1989; Galinski, 1995). Proline transport genes
(proV, proX, proP, proW) in both gram positive and gram-negative bac­
teria encodes MFS-like efflux pump, which export proline and glycine
betaine. The evidence showed representative upregulation of these
genes during the initial stages of biofilm formation (Mauter et al., 2013;
Resch et al., 2005). The same result has obtained in S. epidermis where
SE0225, a glycine/betaine transporter opuCD encoding gene, revealed
almost 3 fold enhanced expression in biofilm cells in compare with
planktonic cells (Yao et al., 2005). In Enteropathogenic E. coli (EPEC), it
has been shown that overexpression of the OmpX protein significantly
restored biofilm formation and curli production under sucrose- and
NaCl- induced hyper-osmotic conditions in ΔtolC; that previously lacked
these properties (Li et al., 2018). Overexpression of OmpX in this

Table 2
Summary of likely efflux pumps involved in early stage of biofilm.
Efflux pump/ Class of pump Bacterial species Potential substrate/Role Main function
Component

Physico-chemical Hydrophobicity CusABCF CusA (RND),CusB Gram -bacteria Cu/Ag Regulation of the
interactions/ Pili and (MFP), CusC hydrophobicity
surface proteins (OMF) Surface positive charge
TolC RND & MFS Enteroaggregative E. coli 38-KD protein Aggregation
(EAEC) (hydrophobic surface in aqueous environment
protein) secretion Aggregation/Adhesion to
AAF regulation host cells in aqueous
environment
Osmoadaptation Glycine betaine/ ABC transporter Gram – and + bacteria Proline and glycine betaine Control hyper-osmotic
L-proline conditions
transporter
Osmoregulation OmpX – Extraintestinal pathogenic NaCl or Sucrose Curli production under
Escherichia coli (ExPEC) transporter regulation hyper-osmotic conditions
TolC RND & MFS Enteroaggregative E. coli Mg2 transporter regulation Adherence
(EAEC) Curli (csgAB) regulation Adherence to abiotic
S. enterica serovar surfaces under osmotic
Typhimurium condition
Pili production AdeABC & RND A. baumannii Expression of type I pilus Adherence to biotic and
AdeIJK MFS A. baumannii ATCC17978 system abiotic surfaces
A1S_0116 Staphylococcus aureus Ac-505 (a lipopeptide-like Adhesion factor
Tet38 compound) Attachment and
Interacting with CD36 internalization into Eps
Motility MexEF-OprN RND P. aeruginosa PA14 PQS Swarming
MexGHI-opmD ⸗ P. aeruginosa PAO1 ⸗
AcrAB-TolC Salmonella Typhimurium chewy, flgLMK and type III
secretion system
regulation/ fumarate
Gene regulation Lm. G_1771 ABC Listeria monocytogenes Dlt, SrtA & GntR regulation Biofilm formation
SmeGH RND Stenotrophomonas negatively repressor
maltophilia Biofilm formation
triggering compounds

8
M.N. Hajiagha and H.S. Kafil
situation may alter the production of NaCl or sucrose transport related
proteins or signal pathways, increasing or decreasing NaCl or sucrose
efflux or influx through the outer membrane.
4.1.2. Pili and surface proteins
The bacterial TolC homologues is a multi-functional protein that
operate many different functions (Koronakis et al., 2004). Enter­
oaggregative E. coli (EAEC)’s major properties are its ability to attach to
Hep-2 cells in vitro and to produce biofilm in the intestinal mucosa
(Wakimoto et al., 2004). Several factors, such as AAF, AggR as the
aggregative factor, and Aap (dispersin) as an anti-aggregative factor
exported by localized outer membrane AatA (homologous to TolC), are
involved in adhesion and biofilm formation of EAEC (Harrington et al.,
2006; Sheikh et al., 2001). In a study conducted by the Naoko et al and
colleagues on EAEC04 showed less aggregation and adherence for TolC-
deleted mutants (042tolC) than the wild type (Imuta et al., 2008). The
loss of the hydrophobicity of the bacterial surface and the decrease in
transcription of the aggregative fimbriae (AAF) are two putative
mechanisms by which the TolC mutant lost the aggregative phenotype.
Another hypothesis that may be implicated in these phenomena is a
disturbance in the regulation of Mg2 transport, which is most probably
correlated to TolC. According to one study, Mg2 is essential for the
adhesion of some EAEC strains (Chart et al., 1997). Moreover, there is a
38-kDa protein (hydrophobic surface protein layer) reported previously
for EAEC (Imuta et al., 2008). It is thought to be a humoral factor
secreted by TolC and unlike Aap, functions as an aggregation factor.
A similar result was reported for EPEC TolC-mutant strains as well as
for wild-type in M9 medium after the administration of efflux pump
inhibitors. They also reported reduced curli production and diminished
csgB and csgD expression (Hou et al., 2014; Li et al., 2018). The curli
proteins aid in the adherence of certain bacteria to abiotic surfaces as
well as host cells (Kim et al., 2012; Prigent-Combaret et al., 2000; Tursi
et al., 2020). The csgBA operon synthesizes the main and minor curli
subunits. These results indicate the ability of TolC to respond to different
osmotic conditions and regulate biofilm formation based on it. Besides,
low expressed level of curli component coding genes (csgB and csgD) was
seen for S. enterica serovar Typhimurium mutants lacking of Acr-TolC
and after using the efflux inhibitor in LB broth without the salt (Baugh
et al., 2012). Curli is a crucial component of the Salmonella extracellular
matrix, and its absence reduces biofilm formation significantly (Baugh
et al., 2012). The exact role of the efflux pump is not recognized, but
some studies have suggested special role by exporting the global regu­
lator as activator on the transcriptional level or can be due to post-
transcriptional changes. Other investigators attribute these effect to
post-transcriptional changes (Baugh et al., 2012).
The AdeABC and AdeIJK pumps in A. baumannii appeared to be
involved in the expression of genes encoding CsuA / B, CsuC, and FimA
proteins (type 1 pilus system) by a similar mechanism with Salmonella as
we described above (Yoon et al., 2015). Eun et al., reported overex­
pressed adeABC and adeIJK mutants show lower biofilm formation due
to lower expression of type I pilus system-related genes (Yoon et al.,
2015). Abiotic surface attachment, colonization, and the formation of
microclonies on abiotic surface can be mediated by these proteins.
Forthermore, DABA-AT that involved in biosynthesis of DAP reduces in
overexpressing adeABC and adeIJK mutants. DAP is a polyamine
required for A. baumannii surface-associated motility. So, the reduction
in DAP production contributes in less motility and biofilm formation.
However, it has not yet been proven that DABA-AT is the substrate of
these pumps.
Different A. baumannii adeABC-deleted strains show different ability
in biofilm formation on different surfaces (Richmond et al., 2016). In
AYE mutants (adeB-deleted A. baumannii), the ability to form biofilms on
both mucosal and plastic surfaces is impaired, but in S1 mutants (adeAB-
deleted A. baumannii), only biofilm formation on mucosal surfaces is
reduced. LIVE/DEAD staining and confocal laser scanning microscopy
on adherent AdeB AYE mutants on mucosal tissue revealed no
9
Infection, Genetics and Evolution 112 (2023) 105459
substantial difference in initial attachment. AdeB-deficient mutants also
have higher levels of pil and com (competence) gene expression. In some
strains downregulation of AdeABC efflux pump, so increased pili pro­
duction may be associated with the ability of A. baumannii to form a
biofilm on plastic. However, these pumps have an important function in
EPs and mature biofilm formation on biotic surfaces in the other
A. baumannii isolates (Richmond et al., 2016).
QS-regulated operon A1S_0112–A1S_0119 contains of A1S_0116
gene which encodes an RND efflux pump in A. baumannii ATCC17978
strain. It’s believed that it’s implicated in the secretion of signaling
molecules or secondary metabolites. Recently, the A1S_0114 gene was
reported within the same operon which produces Ac-505 (a lipopeptide-
like compound named acinetin505) (Rumbo-Feal et al., 2017).
A1S_0116 involved in the secretion of Ac-505 as a secondary metabolite.
The exact role of this pump and Ac-505 in early stage of biofilm pro­
duction hasn’t revealed yet, but deficiency of A1S_0116 may result in
surface motility defect (Blaschke et al., 2021). Tet38 in Staphylococcus
aureus has been proposed as an adhesion factor that facilitates epithelial
cell attachment and internalization by interacting with the scavenger
receptor CD36 (Truong-Bolduc et al., 2021).
4.1.3. Motility
Cell surface Motility and its regulation in some motile bacteria like
Bacillus, Pseudomonas, Vibrio, and Escherichia are required for biofilm
formation. Flagella and motility are probably important early in bacte­
rial adhesion to locate and interact with a suitable surface. Nevertheless,
in the growing and mature stages of biofilm, functional inhibition of
motility aids in the maintenance of the bacterial aggregation (Gutten­
plan and Kearns, 2013). There are 3 types of motilities including
swimming, swarming, and twitching which involve in biofilm formation
(Harshey, 2003). Swimming and swarming are related to flagella
biosynthesis, rotation, and chemotaxis while twitching motility is
dependent on type IV pili (Aybey et al., 2021). Swarming motility is
often influenced by quarum sensing (Partridge and Harshey, 2013), so
we can expect to see inhibition of quorum sensing responses in places
where decreased motility is helpful for biofilm formation; less motile
strain of P. aeruginosa is more competent for biofilm formation within
the cystic fibrosis (CF) lung (Ramsey and Whiteley, 2004).
Martin et al., showed nfxC-type mutants of P. aeruginosa strain PA14
(overexpressing MexEF-OprN) had deficiencies in swarming and biofilm
formation in Tryptic Soy Broth (TSB) due to rapidly pumped of the
Pseudomonas Quinolone Signal (PQS) Precursor HHQ (4-hydroxy-2-
heptylquinoline) (Lamarche and Déziel, 2011). They also observed
knocking out the mexE (the membrane fusion) gene in the MGL01 (a
P. aeruginosa mexS- mutant overexpressing MexEFOprN) restores
swarming motility, pyocyanin production and biofilm formation to wild-
type in vitro. Surveys showed an increased Pseudomonas quinolone
signal (PQS) (one of the AIs in QS system) production due to the efflux of
4-hydroxy-2-heptylquinoline in this strain. This suggests that the
MexEF-OprN efflux pump regulates motility and biofilm formation
through secretion of a QS system signaling molecule (Lamarche and
Déziel, 2011). It needs to compare mentioned phenotypes in various
culture media since it’s been reported an overexpression of the mexEF-
oprN operon near to the primary normal human airways epithelial cells
(Frisk et al., 2004) which represents CF condition. however In another
study the mexEF mutants exhibited enhanced swarming, rhamnolipid
synthesis, and lethality in a mouse infection mode (Vaillancourt et al.,
2021). A gene cluster has been characterized in P. aeruginosa that en­
codes MexGHI-opmD pump. Allelic exchange mutants of mexI and opmD
in P. aeruginosa PAO1 demonstrated poor swarming motility in addition
to other QS-related phenotypes (Aendekerk et al., 2002), but biofilm
formation hasn’t studied yet.
Mutants lacking a component of AcrAB-TolC in Salmonella Typhi­
murium represented changed motility and adherence ability. It’s been
reported less motility in L110 (acrB::aph) than wild-type (SL1344).
Lacking acrA, acrB and particularly tolC gene resulted in poorly adhere to
M.N. Hajiagha and H.S. Kafil
cells in tissue culture and grow in in anaerobic conditions. By using the
Reverse transcription-PCR and Western blotting, they confirmed
decreased expression of numerous genes encoding cheWY and flgLMK
and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secre­
tion system genes, including sopE, and associated effector proteins.
However, acrA, acrB, or tolC can be involved in a regulatory network
that influence in genes that encode proteins involved in this phenomena
(Webber et al., 2009).
Buckner et al., reported that disruption of acrD in S. enterica serovar
Typhimurium led to motility defects caused by changes in intracellular
concentrations of fumarate (Buckner et al., 2016). According to the
distinct study, S. enterica AcrD mutants were incapable to create a
competent biofilm in the crystal violet model (Baugh et al., 2012);
however O-antigen mutants, which are impaired in swarming, were
generally more effective in biofilm production than the wild type
(Mireles et al., 2001). They conclude that inhibition of motility is likely
to be advantageous to prevent bacterial cell dispersal in vitro, but
impaired EPs secretion following AcrD inhibition impairs biofilm evo­
lution in later stages.
As well as reduced swimming and swarming motilities has been
observed in ΔmexB, ΔmexF and mexA mutant in Pseudomonas syringae pv.
tabaci 6605. ΔmexA had fewer flagella than wild type (Ichinose et al.,
2020).
4.1.4. Indirect regulation of biofilm-related genes
Although there are only few reports about how efflux pumps
contribute in biofilm regulation, signal molecule transport by them may
be considered as a part of this role. Zhu et al. (2008) reported a putative
ABC transporter permease encoded by Lm.G 1771 gene in Listeria mon­
ocytogenes that its deleted mutant exhibited an enhanced ability for
biofilm formation compared with WT. Proteomic and transcriptomic
analyses in lm.G_1771 deletion mutant (D1771) showed several cell
surface protein encoding genes may influence by lm.G_1771 (Zhu et al.,
2011). They hypothesized that through a new signal transduction
pathway the lm. G_1771 negatively regulates the expression of several
candidate encoding genes. For example, cell surface proteins (Dlt), cell
surface anchor proteins (SrtA), and transcriptional regulators (GntR)
that involve in biofilm formation. A similar phenotype was observed in
SmeH (the transporter protein of the SmeGH, a RND efflux pump)-
deficient Stenotrophomonas maltophilia mutants. This strain showed a
significantly increased ability to form biofilms, but no difference in
swimming motility was observed compared to the wild strain D457.
Similar to lm. G_1771, this pump shows a negative effect on the regu­
lation of genes involved in biofilm development. In addition, the accu­
mulation of toxic substances following the inactivation of transporter
can trigger a stress respond and subsequently induce the expression of
biofilm genes.
4.2. Effect on growth and maturation
4.2.1. Quorum sensing
During the growth and maturation stages, the accumulation of multi-
layered clusters of microbial cells leads to a series of specially controlled
activities strongly influenced by the genetic and QS control system
(Kjelleberg and Molin, 2002). QS is an intercellular communication
process that allows bacteria to live as multicellular organisms by sensing
their environment and regulating their density and behavior (Bassler
and Losick, 2006). This system involves cell-to-cell communication
based on the synthesis, release, recognition, and response to extracel­
lular signaling molecules known as autoinducers at the group level
(Papenfort and Bassler, 2016). There are 3 main QS-related autoinducers
consist of Acyl-Homoserine Lactones (AHL) in gram negative bacteria,
autoinducing peptides (AIP) in gram positive bacteria and AI-2 as the
inter-species signaling molecules (Wu and Luo, 2021).
There is an interrelationship between efflux pumps and quorum
sensing systems. They arise from each other at the same time, and that
10
Infection, Genetics and Evolution 112 (2023) 105459
interference with one will upregulate or downregulate the other. Efflux
pumps could potentially be involved in quorum sensing and its-related
biofilm formation. This involvement performed by influencing the
transmission of signaling molecules, the effect on the expression of genes
involved in quorum sensing, and antimicrobial resistance mechanism
(Lekshmi et al., 2018). In addition, efflux pumps can help in biofilm
formation by exporting quorum sensing signals produced as inhibitors of
the growth of competing bacteria or noxious QS signals. Besides they
may act as a defense system against quorum sensing inhibitors or
improper signals from other species. The transporters which involved in
QS transportation across the bacterial cell membrane has been listed in
Table 3.
This feature was first reported in P. aeruginosa, where the formation
of a controlled biofilm by quorum sensing is well known. It’s been
claimed that the MexAB-OprM extrude the autoinducer N-(3-oxodode­
canoyl)-L-homoserine lactone (3-oxo-C12-HSL) (Pearson et al., 1999),
and MexEF-OprN has role in 4-hydroxy-2-heptylquinoline extrusion
(HHQ) and kynurenine, as a precursor of HHQ (Alcalde-Rico et al.,
2016). MexB deleted mutants showed an increased QS response due to
the accumulation of acyl-HSLs and its selective binding to LasR (QS
receptor protein) (Minagawa et al., 2012). Several studies have reported
decreased expression of virulence factors regulated by QS following
overexpression of MexAB / OprM and MexEF-OprN efflux pumps
(Alcalde-Rico et al., 2020a; Lamarche and Déziel, 2011; Rasamiravaka
and El Jaziri, 2016). A similar result has been reported for the MexCD-
OprJ efflux pump (Alcalde-Rico et al., 2018). They believe that the
release of intracellular autoinducers by the mentioned pumps is the
cause of this phenomenon, although this theory has recently been
controversial (Alcalde-Rico et al., 2020b).
In Acinetobacter Spp, QS also plays an important role in the formation
and maturation of biofilms. Decreased expression and production of
AHL in A. baumannii leads to a reduction in biofilm formation. The
secretion of long-chain AHLs into the extracellular space is mediated
through efflux pumps. In the review of several clinically isolated
A. baumannii strains, it has been observed that hyper expression of the
AdeFGH efflux pump, especially the upregulation of abaI (an AHL syn­
thase) and abeG (a component of the AdeFGH efflux pump) genes induce
the biofilm formation (He et al., 2015). Nevertheless, In another study,
overexpression of the AdeABC, AdeFGH and AdeIJK pumps has reduced
biofilm formation (Yoon et al., 2015). Generally, in-depth function of
the AcrAB efflux pump in different stages of biofilm formation by QS
hasn’t characterized yet. It may lead to the uncontrolled release of AHLs
outward and a decrease in its intracellular concentration. In
A. nosocomialis, knocking out of the acrA and/or acrB genes resulted in
reduced biofilm/pellicle development and antimicrobial resistance.
Probably it’s due to a disorder in the formation of the extracellular
matrix, as no change in secretion of AHLs was observed (Subhadra et al.,
2020). acrAB mutants may use different efflux pumps for extrusion as a
compensatory instead of AcrAB efflux pump. EMSAs confirmed limited
AHL secretion in the acrR deleted mutant of A. nosocomialis. According
to an in-silico analysis, AcrR may bind to motifs in the promoter regions
of anoI (encoding AHL synthase) and anoR at a higher level. Even
removing it may have caused the overexpression of pumps.
In previous studies it’s been showed that the RND efflux pump
BpeAB-OprB in Burkholderia pseudomallei KHW may be involved in
synthesis or secretion of N-octanoyl-homoserine lactone (C8HSL) (Chan
and Chua, 2005). Likewise, Acinetobacter, it seems AHLs regulate
BpeAB-OprB efflux pump in B. pseudomallei KHW. The adding of C8HSL
and C10HSL autoinducers exogenously promotes bpeAB promoter-lacZ
expression to the early exponential phase (Chan and Chua, 2005).
However Takehiko et al., obtained the opposite results in B. pseudomallei
1026b BpeAB-OprB mutants (Mima and Schweizer, 2010). They found
no defects in AHLs secretion or biofilm development unless they were
accompanied by deficiencies in AmrAB-OprA. Additionally, the Bacter­
oides fragilis BmeB efflux pump establishes a correlation between QS and
biofilm formation by regulating the internal concentration of HSLs
M.N. Hajiagha and H.S. Kafil Infection, Genetics and Evolution 112 (2023) 105459

Table 3
Summary of potential efflux pump involved in growth and maturation stages of biofilm.
Efflux pump/ Class of pump Bacterial species Potential substrate/Role Main function
Component

Quorum sensing MexAB-OprM RND P. aeruginosa 3-oxo-C12-HSL AHL

MexCD-OprJ P. aeruginosa HHQ & kynurenine
MexEF-OprN A. baumannii (OH-dDHL)
AdeFGH B. pseudomallei KHW C8HSL
AdeIJK
AdeABC
BpeAB-OprB
MsrA ABC Staphylococcus spp - AIP
transporter
Secondary AcrEF RND E. coli Indole Signaling molecules in low
metabolites Mtr transporter RND E. coli Nitrosyl indole derivatives concentrations
MdtEF RND Salmonella spp Diamide, H2O2, Paraquat, ADBAC, detergents, toxic in high concentrations
MdsABC RND P. aeruginosa gold, dyes, SDS, novobiocin Toxic in anaerobic respiration and
MexEF-OprN RND E. coli, Salmonella and Phenazines stress conditions
MexGHI-OpmD Shigella Bile salts Toxic metabolites in stress
AcrAB conditions
Iron acquisition, signal molecule &
electron mediator
Toxic in high concentrations
Biofilm inducer in low
concentrations
MexCD-OprJ,
MexCD-OprN RND P. aeruginosa Kynurenine Precursor of anthranilate & AQs
MexEF-OprN SMR E. coli K-12 Spermidine Swarming & antibiotic resistance
MdtIJ RND Gram negative bacteria Cu/Ag Biofilm formation stimulator at low
CusABCF MFS S. aureus Siderophore concentrations
NorA RND P. aeruginosa Siderophore Toxic in high concentrations
MexXY-OprM RND B. pseudomallei KHW Siderophore production Toxic heavy metal
MexAB-OprM RND E. coli & S. enterica PPIX Iron acquisition
BpeAB-OprB A unique E. coli Nickel Iron acquisition
MacAB family S. aureus QAC Iron acquisition
RcnAB MFS E. coli pHBA Haem homeostasis
MdeA ND E. coli Non-metabolic carbohydrates Curli production in low
NorB MFS concentrations
NorC Toxic in high concentrations
AaeAB Biofilm formation stimulator at low
SetAB concentrations
Toxic in high concentrations
Toxic in high concentrations
Toxic in high concentrations

(Pumbwe et al., 2008).
In most Gram-positive QS bacteria AIP is processed and released by
ABC transporters (Bassler, 1999). The MsrA efflux pump is involved in
the expression of the Agr locus (a Gram-positive two-component QS
system). On the other hand, the agr locus regulates biofilm formation in
Staphylococcus spp. Using of baicalin as a MsrA efflux pump inhibitor
reduced transcription levels of agrA, agrC, RNAIII, and sarA (quorum-
sensing system regulators) and the formation of biofilm (Wang et al.,
2019a).
AI-2 is transferred by LsrC, LsrD, and the lsrA (ATPbinding protein),
but the exact mechanism isn’t characterized yet. The lsr operon is
regulated by LsrR as a repressor protein. As well as it’s been determined
that MdtH, an MSF multidrug efflux pump in E. coli, regulated by LsrR. It
needs to assay relationship between AI2/LsrR and MdtH, and their
impact on biofilm formation (Yu et al., 2020).
Rahmati introduces a model in which the actual role of drug efflux
pumps is extruding QS molecules that results in their over production in
response to cell density. According to this, SdiA (suppressor of division
inhibition) seems to control AcrAB efflux pump production positively to
mediate cell–cell communication (Rahmati et al., 2002). Recently, a
novel transcriptional regulator called AnoR has been identified in in
A. nosocomialis which has crucial role in AHL production (Subhadra
et al., 2020). In addition, reduction of surface motility and biofilm for­
mation were also reported in its deleted mutants. The study of these
strains showed a decrease in the expression of acrA and acrB genes,
which indicates the effects of AHLs in the expression of AcrAB multidrug
efflux system. Moreover, LuxS/AI-2 quorum-sensing system in
Streptococcus. suis is involved in efflux pump SatAB regulation (Wang
et al., 2019c). It’s been suggested that determined fluoroquinolones
susceptibility in ΔluxS strains is due to overexpression of efflux pump
SatAB (Wang et al., 2019c).
As a result, incorrect antibiotic dose can not only fail to eradicate the
pathogen, but also aid in the development of biofilms by triggering the
expression of QS- and efflux pump-related genes. In a study by Dawan
et al. On S. Typhimurium strains, this result was clearly observed (Dawan
et al., 2022). S. Typhimurium ATCC 19585 (STWT) and S. Typhimurium
CCARM 8009 (STCI) strains that were only exposed to sub-MICs of CEF,
CHL, CIP, ERY, NOR and TET antibiotics showed a high ability to form
biofilms, while the using of these antibiotics in combination with PAβN
(an efflux pump inhibitor) exhibited significantly decreased the biofilm-
formation.
4.2.2. Secondary metabolites
Multidrug efflux pumps can help in maintenance of homeostatic
levels of biofilm metabolites by extruding the exogenous components
including produced toxic compounds by competitors, heavy metals, and
organic pollutants and endogenous metabolites (Table 3). The precise
function of metabolites, whether they act as hazardous or beneficial
external agents in the formation of biofilms, depends on their intrinsic
properties or their intra- and extracellular concentration. Besides the
location of biofilm within the body, the developmental stage of it, and
the anatomical location of bacterial cells within the biofilm structure can
be as determinants of metabolites function. In the microbial population
enclosed in the biofilm matrix, the oxygen concentration is gradually

11
M.N. Hajiagha and H.S. Kafil
reduced from top to bottom. With anaerobic conditions in the biofilm
center, either anaerobic bacteria predominate or optional anaerobic
bacteria activate the anaerobic respiration pathway for energy.
4.2.2.1. Indole. A tryptophan metabolite synthesized by gut bacteria,
functions as metabolites-mediated QS signal and is involved in biofilm
formation in a different ways (Lee and Lee, 2010). Decreased extracel­
lular secretion of indole or increased intracellular concentration of its
harmful metabolic byproduct following the elimination of their exporter
may disrupt biofilm formation (Kim and Park, 2015).
The AcrEF-TolC and Mtr transporter proteins are reported to be
responsible in the export and import of indole in E. coli (Heatwole and
Somerville, 1991). The mtr gene-deleted mutants showed growth defi­
cient on tryptophan-free medium, which is non-inducible by adding the
external indole (Yanofsky et al., 1991). In the acrEF mutant, there was an
increase in intracellular indole concentration and a decrease in indole
excretion (Kawamura-Sato et al., 1999). Furthermore, in another study,
the use of exopolysaccharide secreted by Lactobacillus plantarum and
Bacillus spp. against E. coli resulted in inhibition of biofilm formation
and efflux pumps (Mahdhi et al., 2018). This phenomenon may be
related to the ability of EPS to alter the physiology of the bacterial
membrane or to reduce indole production and secretion (Mahdhi et al.,
2018). Other studies, however, refuse the protein-dependent transfer of
indole and tryptophan. They claimed that indole transfer across the lipid
layer directly (Piñero-Fernandez et al., 2011; Zarkan et al., 2020). There
are some evidences that indicated some efflux pumps is regulated by
indole; for example the expression of AcrAB multidrug efflux pump in
S. enterica serovar Typhimurium and the expression of mdtABC and acrD
in E. coli are mediated by indole (Hirakawa et al., 2005; Nikaido et al.,
2011).
Under respiratory stress RND efflux pump, MdtEF, extrudes indole
nitrosative derivatives, which are produced during anaerobic respira­
tion of nitrate (Zhang et al., 2011). It has been shown that expression of
MdtEF in E. coli upregulated more than 20-fold anaerobically (Deng
et al., 2013). It is unlikely that the expression of this pump in the center
of the biofilm, where anaerobic conditions prevail, will act as an
exporter of these compounds due to anaerobic respiration. It is necessary
to study the expression of this pump in biofilm members in the presence
or absence of antibiotics in order to discover its physiological signifi­
cance. MdsABC, a RND efflux pump, in Salmonella also protects biofilm
cells from stressful conditions due to its wide range of substrates such as
diamide, hydrogen peroxide and Paraquat, alkyldimethylbenzylammo­
nium chloride (ADBAC), detergents, gold, dyes, SDS and novobiocin
(Baugh et al., 2012; Sun et al., 2014). Decreased ability to form biofilms
has already been observed in mutants without this pump (Baugh et al.,
2012).
4.2.2.2. Phenazines. The effect of phenazines in P. aeruginosa on biofilm
formation varies from beneficial (as signal molecules) to harmful
(generating toxic superoxide species). Two samples of phenazine com­
pounds, phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO), have
been studied for their effect on biofilm formation. PCA enhances biofilm
formation in P. aeruginosa mutant lacking the ability to produce the
siderophores pyoverdine and pyochelin, by iron acquisition through the
reduction of Fe (III) to Fe (II) (Wang et al., 2011). In contrast to PCA,
pyocyanin (PYO) may promote biofilm development through a different
mechanism. It is a virulence factor that acts as a signal molecule in
addition to being an electron mediator. 5-methylphenazine-1-carbox­
ylate (5-Me-PCA) another intermediate compound with high redox po­
tential has been introduced recently, which is involved in conversion of
PCA to pyocyanin. It has been revealed to be essential for biofilm for­
mation because it promotes extracellular electron transfer in the biofilm
(Sakhtah et al., 2016). In P. aeruginosa, MexEF-OprN and MexGHI-
OpmD pumps, in addition to the effect on fluoroquinolone resistance,
are involved in controlling the concentrations of both toxic and non-
12
Infection, Genetics and Evolution 112 (2023) 105459
toxic phenazines. Especially the steady-state in the production and
release of phenazines is controlled by MexG/HI-OpmD (Huang et al.,
2018; Jin et al., 2015; Sakhtah et al., 2016; Wolloscheck et al., 2018). In
a study conducted by the author and his colleagues, this issue was
examined in detail and the results obtained confirmed the above
mechanism (Wolloscheck et al., 2018). They observed a significant
decrease in extracellular pyocyanin so that the concentration of it in
culture medium supernatant was measured almost 2-fold lower
compared to the wild strain. Also, the high amount of pigment in the
culture of PAO1 (overexpressing MexGHI-OpmD or MexHI-OpmD
mutant) indicated the role of this pump in pyocyanin production.
Conversely, these phenazines also induce upregulation the MexGHI-
OpmD expression by a SoxR (transcription factor)- dependent mecha­
nism (Dietrich et al., 2006; Du et al., 2015; Sakhtah et al., 2016).
4.2.2.3. Bile salts. A group of bactericidal exogenous metabolite which
are synthesized in the liver and presence in the duodenum, jejunum, and
proximal ileum. However, Bile is lethal at high concentrations induces
expression of virulence genes at low concentrations, including efflux
pumps. Intestinal bacteria are resistant to these compounds by using
multiple strategies. Since bile salts are considered as one of the sub­
strates of efflux pumps, it seems that exporters protect the bacteria
against the toxic amounts of bile salts. The AcrAB efflux pump is one of
the most crucial RND families in E. coli, Salmonella and Shigella is used to
resist bile salts (Buckley et al., 2006; Nickerson et al., 2017; Nikaido and
Zgurskaya, 2001). ΔacrB mutant in Shigella was unable to growth in
0.4% and 2% bile salts (Nickerson et al., 2017). As well as in Salmonella
the acrA, acrB, and tolC mutants could not adapt to bile (Urdaneta and
Casadesús, 2018). However, in both bacteria, long-term incubation with
sub-inhibitory concentrations has been shown to induce the expression
of these pumps. Quantitative reverse-transcription (qRT)-PCR analysis
revealed that acrB and some others genes including S2558, ospE1-ospE2
and yhcN of Shigella were induced almost 2-fold in prolonged exposure
to 0.4% bile salts (Nickerson et al., 2017). In a similar investigation the
expression level of the acrA and acrB genes in the presence of pre-
inhibitory amount of DOC was obtained approximately 10 fold during
exponential growth (Urdaneta and Casadesús, 2018). The AcrAB-TolC
efflux pump in Salmonella is regulated by ramA (an efflux transcrip­
tional regulator). Sylvie et al identified the bile-mediated activation of
acrB and tolC proceed in a in a ramA-dependent manner (Baucheron
et al., 2014). Bile specifically binds to RamR (a repressor of ramA) and
transcriptionally actives ramA (Ricci et al., 2012).Not only in these two
bacteria but also in several intestinal pathogens such a bile salt-
mediated transcription of efflux pump has been reported (Nishino
et al., 2009).
It has also been shown that long-term exposure to bile increases
biofilm formation in some bacterial species like Clostridium difficile,
Vibrio cholera and Shigella flexneri that is known to enteric bile salt-
induced biofilm (Dubois et al., 2019; Hung et al., 2006; Nickerson and
Faherty, 2018). This is a transient phenomenon that prepares a rapid
dispersion opportunity to bacteria to colonize in the epithelial cell sur­
faces in the small intestine and then reside in the colon where includes
low concentrations of the bile (Gholizadeh et al., 2019; Gupta et al.,
2016). The VexAB pump of V. cholerae, the CmeABC Pump in
Campylobacter jejuni, the HefC Efflux Pump in Helicobacter pylori,
EmrAB of the MFS in E. coli are the other examples of efflux pump which
are contributed in protection of toxic bile metabolites. However, a
definitive association between overexpression of the pump and bile-
induced biofilm has not yet been investigated. Nevertheless,
increasing multidrug resistance or enhances the pump of substrates
involved in the extracellular component can be suggested as its func­
tional role in biofilm development. Furthermore, by removing the
inducing amounts of bile salts, it will lead to more dispersion in the
lower parts of the gastrointestinal tract. Biofilm formation in efflux
pump mutants in the presence of inducing level of bile can assist to
M.N. Hajiagha and H.S. Kafil
elucidate this topic substantially.
4.2.2.4. Kynurenine. An intermediate metabolite in the tryptophan
degradation pathway, is used as a precursor of anthranilate and alkyl-
quinolone signal (AQs) molecules. This metabolite is also associated
with some phenotypes like swarming, antibiotic resistance (Butt et al.,
2016). Results revealed that kynurenine extrusion is mediated by
MexCD-OprJ and MexCD-OprN in the P. aeruginosa which their over­
expression lead to an impaired QS response (Alcalde-Rico et al., 2018)
and biofilm formation (Favre-Bonté et al., 2003). Despite the existence
of a wide range of different substrates for these pumps and other reasons
for justifying this result, the author believed that this impairment is
mainly due to excessive removal of kynurenine as a precursor of HHQ in
the MexEF-OprN overexpressing mutant (Alcalde-Rico et al., 2018;
Olivares et al., 2012).
4.2.2.5. Spermidine. Spermidine is a polyamine compound that found
in many bacterial cells. It has role in various cellular processes like
regulation of biofilm formation (Thongbhubate et al., 2021), but the
overaccumulation of this polyamine secondary metabolite can cause the
cell toxicity (Fukuchi et al., 1995). MdtIJ belongs to SMR superfamily is
responsible for transporting excess spermidine out of the cell (Higashi
et al., 2008). Considering that intracellular spermidine stimulates bio­
film formation, it can be expected that deleting this pump will lead to
increased biofilm formation. An increase in biofilm mass has previously
been reported in mdtJ-deficient mutants in E. coli K-12 (Bay et al., 2017).
However, Thongbhubate and colleagues reported no difference in the
capacity of the wild type and deleted-mdtJ strains to achieve this phe­
nomena (Thongbhubate et al., 2021). In another study, even an increase
in the expression of this pump was observed during the growth of the
biofilm (Kvist et al., 2008).
4.2.2.6. Heavy metals
4.2.2.6.1. Copper and silver. Copper and silver are two very toxic
compounds for bacterial cells (Ameh et al., 2022) that have previously
been mentioned for their possible positive effects in the early stages of
biofilm formation. Therefore, reducing their intracellular concentration
by CusABCF pump (Betts, 2020), a heavy metal RND efflux pump (HME-
RND), seems to be vital for bacterial survival and biofilm structure
maintenance.
4.2.2.6.2. Iron. Iron mediates biofilm formation through oxidative
stress, regulating surface motility, stabilizing the polysaccharide matrix,
serving as the signal agent and etc. On the other hand, intracellular
overaccumulation poses iron-mediated oxidative stress that potentially
damage to bacterial cell. Acquisition of iron is mediated by siderophore,
a small, high-affinity iron-chelating compound with high affinity for
extracellular Fe3+. siderophore is secreted by efflux pumps, which in
gram-negative bacteria due to the presence of a dual membranes it needs
to be exported in a multistep process. First both the ABC transporter and
major facilitator superfamily (MFS) transport siderophore through the
inner membrane. Second TolC-like efflux pumps excrete it across the
outer membrane. EntS (MFS-class efflux pump) in E. coli is responsible
for the export of enterobactin (known as strongest siderophore) from the
cytoplasm to the periplasm. Then, enterobactin is transported across the
outer membrane by the coordination of the inner-membrane RND-class
efflux pump (AcrB, AcrD, or MdtABC) with the outer membrane protein
channel TolC (Horiyama and Nishino, 2014). There are various RND
pumps in other bacterial species as well, which are involved in side­
rophore secretion, like the ApeX in Bacillus anthracis for petrobactin,
VexGH in V. cholerae for vibriobactin, and both MmpL4 and MmpL5 in
Mycobacterium tuberculosis for mycobactin and carboxymycobactin.
PvdRT-OpmQ and MdtABC-OpmB in P. aeruginosa for pyoverdine,
MacAB in S. enterica and E. coli for salmochelin and enterotoxin are
examples of ABC siderophore transporters (Liu et al., 2022).
NorA (MFS efflux pump) as a main efflux pump of S. aureus is
13
Infection, Genetics and Evolution 112 (2023) 105459
contributed in siderophore secretion. It has been suggested that the
transcription of this pump is inversely related to the environmental
concentration of the iron and is in fact iron responsive. They showed that
the addition of FeCl3 in chemically defined medium (CDM) inhibited the
expression of the pump while the ferric uptake regulator protein (Fur)
regulated its expression positively (Deng et al., 2012). Decreased biofilm
formation and biofilm maturation in S. aureus according to combina­
tional treatment of nilotinib (as an efflux pump inhibitor) and the flu­
oroquinolone ciprofloxacin indicates importance of this pump in biofilm
production process (Zimmermann et al., 2019).
Another study reported that MexAB-OprM and MexXY is overex­
pressed in P. aeruginosa in iron depletion conditions, implying that these
pumps is critical for P. aeruginosa survival under these conditions (Poole
et al., 1993). BpeAB-OprB was also shown to facilitate the production of
siderophore in B. pseudomallei KHW. bpeAB null mutant and bpeR
(regulator of bpeAB) over expressing strains were impaired in side­
rophore production and biofilm formation (Mima and Schweizer, 2010).
Therefore, the simultaneous use of iron chelators and inhibitors of these
pumps could synergistically prevent the growth and formation of bio­
films (Liu et al., 2010).
Excess haem (iron-containing molecule) acquisition into the bacte­
rial cell will also disrupt haem natural homeostasis, resulting in the
accumulation of an immediate heme precursor called protoporphyrin IX
(PPIX). Bacteria use a pump belonging to the ABC family (MacAB) to
regulate the intracellular concentration of these metabolite, which has
been reported in E. coli and Salmonella (Turlin et al., 2014). Deletion of
this pump in S. enterica has led to a reduction in biofilm formation,
which may also indicate its role in natural haem homeostasis in biofilm
cells (Baugh et al., 2012).
4.2.2.6.3. Nickel. It was confirmed that nickel triggers biofilm for­
mation by inducing the curli expression (csgBA genes) in sub-inhibitory
concentrations in E. coli (Perrin et al., 2009). In the presence of higher
concentrations, RcnAB efflux pump is used to transport the excess
amount of nickel. The mutant ARY023 strain lacking rcnA (yohM) dis­
played significantly more susceptibility to Ni and Co and increased
intracellular accumulation of nickel (Rodrigue et al., 2005).
4.2.2.6.4. Quaternary ammonium compound:. Different types of
multidrug-resistance efflux pumps for the extrusion of quaternary
ammonium compounds have been investigated in both Gram-positive
and Gram-negative bacteria that are also involved in the formation of
biofilms (Buffet-Bataillon et al., 2016). For example, the members of
MFS superfamily MdeA, NorB and NorC encoding genes, which function
as QAC transporter in S. aureus were upregulated in biofilm growth stage
(He and Ahn, 2011). Some studies have demonstrated that the presence
of these compounds in the environment trigger the expression of efflux
pumps and biofilm formation (Buffet-Bataillon et al., 2016). This in­
duction is probably due to the triggering of stress responses following
exposure to QACs, which is also effective in increasing the level of
surviving persisters and dormant cells (Buffet-Bataillon et al., 2016).
Some efflux pumps are particular transporters of a specific metabo­
lite that is upregulated in high amounts to protect the bacterium from
the compound’s harmful metabolites. The administration of p-hydrox­
ybenzoic acid (pHBA) increased the expression of yhcRQP, in E. coli.
pHBA is an aromatic organic acid metabolite produced during the ubi­
quinone production process, which is generally found in small amounts.
Hence they renamed this family to aromatic carboxylic acid efflux
(AaeAB efflux system) as metabolic relief valve to keep bacteria
balanced metabolically (Van Dyk et al., 2004). Another example is SetA
and SetB in E. coli that belongs to MFS family is involved in glucose
extrusion and upregulated during the biofilm maturation. One of the
functions of this pump is most likely to protect bacterial cells from the
accumulation of non-metabolic carbohydrates like IPTG and methyl-β-D-
thiogalactoside (TMG) that are harmful to them (Liu et al., 1999a; Liu
et al., 1999b).
M.N. Hajiagha and H.S. Kafil Infection, Genetics and Evolution 112 (2023) 105459

Table 4
Summary of potential efflux pumps involved in production of biofilm exopolymeric substances (EPS).
Efflux pump/ Class of Bacterial species Potential substrate/Role Main function
Component pump

Exopolymeric Exopolysaccharides SetB MFS E. coli Glucose Biofilm matrix generation

substance (EPS) EmrE SMR E. coli Arabinose Exopolysaccharide matrix
AraE - Salmonella spp Arabinose generation
A1S_1117 - A. baumannii Sugars Repressor role in regulation and
TetA(C) MFS Salmonella & Tetracycline production of curli and c-diGMP
Alg44 - E. coli Associated with the ABC transporter- Biofilm matrix generation
LT42 ABC Pseudomonas dependent EPS assembly system/ Promotion of colanic acid
S. mutants interaction with AlgE transporter (cspE) expression
Glucosyltransferase Synthesis and secretion of alginate
Sucrose-associated colonization in
vivo
Amino acids and YhdWXYZ ABC E. coli L-leucine Stability of the aggregation
peptides YfiK & YdeD E. coli L-cysteine Stability of the aggregation
eDNA PMT MFS A. baumannii eDNA Adherence to abiotic (polystyrene)
AIIMS 7 & E. coli and biotic (S. cerevisiae/HeLa)
surfaces

4.2.3. Biofilm matrix
4.2.3.1. Exopolysaccharides. Sugars, often in the form of poly­
saccharides in combination with other substances, make up a large
portion of the polymer structure. Some of well-known biofilm matrix
sugars are including polyanionic (alginate and colanic acid) and Poly­
cationic (N-acetylglucosamine) exopolysaccharides (Allison, 2003;
Flemming and Wingender, 2010).
The uptake and export of many hexoses like glucose, galactose,
mannose, and fructose in bacteria generally are mediated by sugar efflux
transporter, a group of transporters similar to MFS proteins (Liu et al.,
1999b). It seems the function of these transporters affect the composi­
tion of the biofilm matrix (Pasqua et al., 2019). The previously described
upregulation of SetB pump in E. coli can be expanded to participation in
the biofilm matrix generation by glucose excretion as well (Pasqua et al.,
2019). Arabinose is a 5-carbon monosaccharide that uses AraJ (Reeder
and Schleif, 1991), Cmr (mdfA) (Kredich and Tomkins, 1966), EmrD
(Koita and Rao, 2012) and YeA (Bost et al., 1999) pumps to transmit
through bacterial membranes. The emrE lacking strains showed a
decrease in biofilm formation that some investigations proposed the
contribution of arabinose in exopolysaccharide matrix (Alav et al., 2018;
Matsumura et al., 2011). Nevertheless Erin et al., observed a hyper­
biofilm structure in araE-deleted mutant of Salmonella in the presence of
L-arabinose (Vasicek et al., 2021). Subsequently the addition of arabi­
nose to the culture medium increased the thickness of the biofilm
without causing dispersion. It is probable that arabinose suppresses the
expression and production of curli fimbriae and GMP as two controlling
factors of the extracellular matix. (Vasicek et al., 2021). The biofilm
cells, not the planktonic cells in Acinetobacter, encode the efflux gene
A1S_1117, which belongs to the msf family (Rumbo-Feal et al., 2013).
Several studies have suggested that this pump may be involved in sugar
transport and biofilm matrix formation, but this has not yet been
confirmed (Alav et al., 2018; AlMatar et al., 2021).
Colanic acid in bacteria such as Salmonella and E. coli contribute in
biofilm matrix formation and its encoding genes are upregulated in
during biofilm formation (Prigent-Combaret et al., 1999; Prigent-Com­
baret et al., 2000). It’s been confirmed an overexpression of the tetra­
cycline resistance efflux pump TetA(C) pump in response to osmotic
stress condition. Results showed enhanced expression levels of colanic
acid transporter (cspE) in presence of 50 mM NaCl and antibiotic
(Ampicillin and Tetracycline) in that indicate contribution of high-level
TetA(C) pumps in promotion of colanic acid biosynthesis and biofilm
maturation (May et al., 2009).
Alginates are the most abundant exopolysaccharides in the Pseudo­
monas biofilm matrix in the lungs of cystic fibrosis patients (Hentzer
et al., 2001). Alg44 (membrane-anchored protein) and AlgE (outer
membrane protein) are two component involved in synthesis and
secretion of alginate, respectively (Hay et al., 2010). The periplasmic C
terminus of Alg44 is hypothesized to be homologous to membrane
fusion proteins implicated in periplasmic transforming in efflux pumps
(Remminghorst and Rehm, 2006). Previous studies have reported a
decrease in algE expression in mutants without Alg44 (Oglesby et al.,
2008). Alg44, which is associated with the ABC transporter-dependent
EPS assembly system model and drug efflux pumps, might also
interact with AlgE (Oglesby et al., 2008). In agreement with role of
Alg44 in synthesis and secretion of the exopolysaccharide alginate, the
alginate co-polymerase Alg44 deleted strains produced a thin and low
cohesive biofilm in compared with wild strain (Noirot-Gros et al., 2019;
Whitney et al., 2015).
An ABC transporter (LT42) and a putative multidrug efflux pump
(LT43) have been identified in S. mutants which seem important in
sucrose-associated colonization in vivo. The deleted multidrug efflux
pump mutant was impaired to colonize the rats otherwise had normal
glucosyltransferase activities (Tao and Tanzer, 2002).
4.2.3.2. Amino acids and peptides. Unlike D-amino acids, positively
charged L-amino acids help in the stability of the aggregation in biofilm
by interacting with other biofilm matrix components (Flemming and
Wingender, 2010). YhdWXYZ system, an ABC-type transporter, in E. coli
has role in L-leucine transportation (Zheng et al., 2018). An almost two-
fold increase in yhdX gene expression in biofilm cells has been reported
compared to planktonic cells (Riley et al., 2006). YfiK incorporation
with YdeD protein exported L-cysteine cross the membrane in E. coli
(Franke et al., 2003).
4.2.3.3. Extracellular DNA (eDNA). In some bacteria, such as S. aureus
and A. baumannii, eDNA is one of the primary biofilm matrix compo­
nents, which could lead to a more compressed and stable biofilm. A MFS
transporter-like ORF (PMT) of 453 in a pathogenic strain A. baumannii
AIIMS 7 has been described that may has a role in adherence to abiotic
(polystyrene) and biotic (S. cerevisiae/HeLa) surfaces (Sahu et al.,
2012). The results confirmed a 2.58-fold increase in the production of
eDNA in PMT gene-cloned E. coli in comparison with E. coli harboring a
control plasmid.
There is a list of efflux pumps in Table 4 which have probable role in
secretion or production of exopolymeric substances according to the
studies.
5. Conclusion
Bacterial biofilms have always been one of the dilemmas in the field
of the health-care system, especially in relation to catheters, artificial

14
M.N. Hajiagha and H.S. Kafil
organs and other medical equipment. The biofilm structure possesses its
resistance to drugs and conditions to several mechanisms that make it
difficult and sometimes impossible to overcome with it. Efflux pumps,
which are usually known for their role in multidrug resistance, seem to
be effective in the formation of biofilm structure in addition to drug
extrusion. Based on the studies, it seems that the expression of efflux
pumps is different in various places and stages of biofilm formation
under the influence of their role in interactions, mobility, gene regula­
tion, QS, secretion of EPS, and the secondary metabolites. Therefore, it is
possible that the incorrect use of antibiotics or efflux pump inhibitors,
instead of proper treatment, can induce the expression of a pump
effective in biofilm formation or inhibit a pump with a negative effect on
it, respectively. Therefore, in order to choose the correct combat strat­
egy, it is recommended to carefully examine the role of pumps in biofilm
formation in each bacterium, range of substrates, and their expression
level separately.
Funding
This study was supported by Drug Applied Reserahc Center, Tabriz
University of Medical Sciences as a grant to Dr Hossein Samadi Kafil.
Code availability
Not applicable.
Ethics approval
This study was a literature review and Ethic was not applicable.
Authors’ contributions
H.S.K participated in data collection, annotation, drafting manu­
script, writing and final approve.
M.N.H participated in data collection, annotation, drafting manu­
script, writing and final approve.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Declaration of Competing Interest
None to declare.
Data availability
No data was used for the research described in the article.
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