Ecotoxicology and Environmental Safety 264 (2023) 115389

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Review

Bacterial biofilm inhibitors: An overview

Vipin Chandra Kalia , Sanjay K.S. Patel , Jung-Kul Lee *
Department of Chemical Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea

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

Edited by Professor Bing Yan Bacteria that cause infectious diseases adopt biofilms as one of their most prevalent lifestyles. Biofilms enable
bacteria to tolerate environmental stress and evade antibacterial agents. This bacterial defense mechanism has
Keywords: rendered the use of antibiotics ineffective for the treatment of infectious diseases. However, many highly drug-
Antibiotics resistant microbes have rapidly emerged owing to such treatments. Different signaling mechanisms regulate
Antipathogens
bacterial biofilm formation, including cyclic dinucleotide (c-di-GMP), small non-coding RNAs, and quorum
Biofilm
sensing (QS). A cell density-dependent phenomenon, QS is associated with c-di-GMP (a global messenger), which
Biomolecules
Inhibitors regulates gene expression related to adhesion, extracellular matrix production, the transition from the planktonic
Peptides to biofilm stage, stability, pathogenicity, virulence, and acquisition of nutrients. The article aims to provide
Polysaccharides information on inhibiting biofilm formation and disintegrating mature/preformed biofilms. This treatment en­
Quorum sensing ables antimicrobials to target the free-living/exposed bacterial cells at lower concentrations than those needed to
Healthcare treat bacteria within the biofilm. Therefore, a complementary action of antibiofilm and antimicrobial agents can
be a robust strategic approach to dealing with infectious diseases. Taken together, these molecules have broad
implications for human health.

1. Introduction
Among infectious diseases, the contribution of biofilm-forming
bacteria has gained attention. Biofilm-forming bacteria cause around
65–80% of infectious diseases (Lebeaux et al., 2013). Such
biofilm-related infectious diseases are typically chronic (Bjarnsholt,
2013). Bacteria within the biofilm are challenging to treat with antibi­
otics, leading to prolonged illness and contributing significantly to high
morbidity and mortality rates (WHO, 2014). The global economic
burden due to the emergence of antibiotic-resistant bacteria is estimated
to reach USD 100 trillion by 2050, and the associated death toll will
increase by up to 10 million per year (Gandra et al., 2014; WHO, 2014).
A comprehensive global estimate reveals that infectious bacteria cause
7.7 million deaths, 13.6% of all global deaths. This has drawn the
attention of health authorities to develop strategies to mitigate this
socio-economic burden. The following five types of biofilm-forming
bacteria have been reported to be responsible for 54.9% of these
deaths: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Staphylococcus aureus, and Streptococcus pneumoniae (Donlan, 2001; Tien
et al., 2007; Lallukka et al., 2017; Vestby et al., 2020). Despite the di­
versity of infectious diseases, there was a significant reduction in mor­
tality and morbidity between 2000 and 2020. This change is primarily
due to improved hygiene, higher accessibility to health care, and med­
ical advances, such as vaccines and antimicrobial therapies (Baik et al.,
2022). Nevertheless, the social and economic burden remains substan­
tially higher in lower-middle-income countries due to poor sanitation
and limited access to healthcare (Lallukka et al., 2017; Bloom and
Cadratte, 2019; Baker et al., 2022). Additionally, the emergence of new
infectious diseases and the reemergence of old ones, such as COVID-19,
highlight the ongoing need for vigilance and preparedness in the face of
infectious diseases (Khabbaz et al., 2015; Morens and Fauci, 2020). It is
essential to evaluate the information generated by various studies to
inhibit the different processes leading to biofilm formation and disin­
tegration the mature biofilms (Kannappan et al., 2017a; Koo et al., 2017;
Srinivasan et al., 2018).
Pathogenic bacteria causing various infectious diseases switch be­
tween a planktonic state and biofilm form to tolerate environmental
stress, especially those caused by antimicrobial agents (Roy et al., 2018).

Abbreviations: AHL, acyl homoserine lactone; AI, autoinducer; BP, bacteriophage; c-di-GMP, cyclic dimeric guanosine monophosphate; DNase, deoxyribonuclease;
ECM, extracellular matrix; eDNA, extracellular DNA; EPS, Exopolysaccharide; MRSA, methicillin-resistant Staphylococcus aureus; NP, nanoparticle; QS, quorum
sensing; QSI, QS inhibitor; QSS, QS system; sRNA, small non-coding RNA.
* Corresponding author.
E-mail address: jkrhee@konkuk.ac.kr (J.-K. Lee).

https://doi.org/10.1016/j.ecoenv.2023.115389
Received 18 May 2023; Received in revised form 5 August 2023; Accepted 17 August 2023
Available online 25 August 2023
0147-6513/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
V.C. Kalia et al.
Biofilm-forming bacteria can often evade the body’s immune system,
making fighting these infections even more challenging. Moreover,
biofilms form on medical devices and implants, increasing the risk of
infection during surgeries or other medical procedures (Kannappan
et al., 2017b, 2019a; b; Kannappan et al., 2020). Preventive measures,
such as regular cleaning of medical devices and implants, improved
hygiene practices in healthcare settings, and the development of new
antimicrobial agents, are crucial in preventing the spread of
biofilm-forming bacteria and reducing the incidence of infectious dis­
eases caused by these bacteria. In addition to the pathogens listed above,
the few most frequently encountered bacterial biofilms in healthcare
centers are Acinetobacter spp., Enterococcus faecalis, and Proteus mirabilis.
The most challenging part of these persistently spreading infections is
the enhanced tolerance of bacterial pathogens to biocides. This
enhanced drug resistance is due to many factors, the most important
being the delayed and incomplete penetration of antimicrobials through
the EPM, the transmission of plasmids conferring drug resistance, and
the emergence of persister cells (Borriello et al., 2004; Lewis, 2007;
Stewart et al., 2009). Biofilm protects microorganisms, making it diffi­
cult to eradicate them compared to their free-living/planktonic coun­
terparts. Bacteria within biofilms can tolerate up to 1000-fold higher
antibiotic concentrations in extreme cases than their free-living coun­
terparts (Kalia et al., 2019). Antimicrobial peptides, polyamines, phy­
tochemicals, and nanoparticles (NPs) possess specific features that
enable them to inhibit biofilm formation. At the other end of the spec­
trum are enzymes, bacteriophages (BPs), and NPs, which target mature
biofilms (Kumar et al., 2021). This review deals with the various ap­
proaches employed to control biofilms and their potential to treat
biofilm-based infectious diseases. Briefly, the antibiofilm agents target
the multiple mechanisms and components involved in the biofilm for­
mation, which include: (i) inhibiting initial adhesion, (ii) removal of
mature biofilms and degradation of various components of the biofilm,
(iii) interference with bacterial communication or quorum sensing (QS),
and (iv) inhibiting non-QS regulatory processes..
2. Biofilms
Biofilm formation is a multistep process, which involves (i) bacterial
attachment, (ii) maturation, and (iii) bacterial release (Flemming et al.,
2016). Free-living or planktonic bacterial cells initiate biofilm formation
Fig. 1. Diagrammatic representation of bacterial biofilm formation and dispersal. Antibiofilm agents act against the signaling systems: cyclic dinucleotide, quorum
sensing, and small non-coding RNAs and the gene expressions responsible for adhesion, extra-cellular matrix production, transition from planktonic to biofilm stage,
stability, pathogenicity, virulence, and acquiring nutrients.
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Ecotoxicology and Environmental Safety 264 (2023) 115389
by aggregating in an aqueous environment (Melaugh et al., 2016). These
aggregated bacteria adhere to a solid surface and produce extracellular
polymeric substrates (EPS), which include polysaccharides, proteins,
lipids, and DNA, providing solid support for maintaining the integrity
and survival of the bacteria termed “matrixome” (Claessen et al., 2014;
Coughlan et al., 2016; Kannappan et al., 2019a; Karygianni et al.,
2020a). Their motility, cell surface proteins, adhesins, protein autolysin,
shear, and electrostatic interactions influence the bacterial adherence
process. In the subsequent stage, bacterial cell proliferation, aggrega­
tion, and differentiation form a mature biofilm (Stoodley et al., 2002).
Bacterial cells from the mature biofilm can undergo detachment and
enter another cycle of biofilm formation, resulting in the spread of in­
fections to other parts of the medical devices or organs (Stoodley et al.,
2002; Fey and Olson, 2010). Exopolysaccharides (EPSs) suppress host
immune responses and confer antibiotic resistance to pathogens
(Angelin and Kavitha, 2020; Li et al., 2022). Extracellular proteins that
enhance biofilm formation and stability vary considerably among or­
ganisms. eDNA coordinates bacterial twitching motility, metal chela­
tion, quenching of positively charged antibiotics, and acquisition of
antibiotic resistance genes, contributing to antibiotic resistance, bacte­
rial aggregation, and stabilization. The interaction among eDNA, pro­
teins, EPS, and other molecules enables biofilm formation and structural
integrity (Panlilio and Rice, 2021). The upregulation of genes encoding
ECM metabolism and cell motility precedes bacterial dispersal. Genes
responsible for synthesizing EPS and fimbriae are downregulated
(Armbruster and Parsek, 2018). In the dispersal stage, enzymatic
degradation of EPS disassembles the biofilm matrix, releasing microbes
and expanding infected regions (Fleming and Rumbaugh, 2017).
Genetic regulation of biofilm formation operates primarily through
three primary mechanisms, which are: (i) QS, (ii) cyclic dinucleotide,
and (iii) small non-coding RNAs (sRNAs). A perusal of the various
studies which have been targeting these three regulatory systems to
manipulate biofilm formation reveals that only a few of them have re­
ported the potential of cyclic dinucleotides (López-Baena et al., 2019),
and (ii) small non-coding RNAs (sRNAs) as drug targets (Taylor et al.,
2017; Manfiolli et al., 2018). In contrast, most studies have revealed QS
systems (QSS) as the significant drug targets (Subhadra et al., 2018;
Kalia et al., 2019).
V.C. Kalia et al.
2.1. Cyclic dinucleotide
Bacterial regulatory signaling molecules, such as cyclic dimeric
guanosine monophosphate (c-di-GMP), confer abilities to respond and
adapt to changing environments (López-Baena et al., 2019). This ubiq­
uitous bacterial messenger regulates different processes. The intracel­
lular fluctuation in its concentration contributes to switching from
motile to sessile and biofilm-forming lifestyles (Moscoso et al., 2011;
Pérez-Mendoza et al., 2014). The major virulent factors include type
secretion systems (TSSs) and their associated effector proteins. Some of
these TSS participate in suppressing host immune responses during
infection. Type 1 secretion systems (T1SS) secrete adhesins, which are
critical in binding to the host, Type 3 secretion systems secrete effectors
to suppress host immunity, and c-di-GMP also regulates the Type 6
secretion systems to export ATPase (Tran et al., 2014; Trampari et al.,
2015; Wang et al., 2017; Kan et al., 2018). T1SS secretes adhesins
through the ATP-binding cassette transporter. Protein translocation
occurs via a periplasmic intermediate (Smith et al., 2018). The secretion
of Lap adhesins results in the autophosphorylation of the GacS sensor,
which transmits its phosphate group to transcriptional regulators. This
mechanism triggers a regulatory cascade transcribing many genes.
During the limitation of nutrients, binding occurs between c-di-GMP to
regulatory protein LapD, which induces sequestration of protease LapG.
Under low c-di-GMP concentrations, biofilm dispersal takes place (Yuan
et al., 2015).
Norspermidine, a polyamine secreted by Vibrio cholerae, regulates
biofilm formation. Deleting the nspC gene encoding for the decarbox­
ylase enzyme involved in norspermidine synthesis reduces biofilm for­
mation (Lee et al., 2009). The NspS/MbaA signaling system binds the
NspS protein to norspermidine, inhibiting MbaA phosphodiesterase ac­
tivity. Furthermore, it also enhances c-di-GMP concentration to promote
biofilm formation. Norspermidine within biofilm also assists in
acquiring iron, which helps strengthen biofilms (Wotanis et al., 2017). A
few other works have elucidated that c-di-GMP mediates the production
of the bacterial ECM, leading to biofilm formation (De Smet et al., 2021).
Bacterial binding to the surface during biofilm formation coincides with
the activation of diguanylate cyclase enzymes (DGCs) involved in
C-di-GMP synthesis. It results in loss of motility due to DGC (SadC) and
an increase in exopolysaccharide production due to another DGC (RoeA)
(Merritt et al., 2007, 2010; Christen et al., 2010; Massie et al., 2012).
cAMP levels increase from low in planktonic cells to high after a few
rounds of bacterial attachment/detachment. As this process progresses,
irreversibly attached cells transform into strong biofilm structures
(Armbruster and Parsek, 2018). C-di-GMP is degraded by the phospho­
diesterases (PDEs) DipA and RbdA (Basu and Sauer, 2014; Petrova et al.,
2015). Finally, c-di-GMP is reported to be inducing biofilms’ dispersal to
initiate subsequent rounds of bacterial infection (De Smet et al., 2021).
2.2. Small non-coding RNAs
Multiple mechanisms and pathways regulate biofilm formation. A
signal transduction system mediates microbial adhesion and ECM syn­
thesis within biofilms through various transcription factors. In addition
to transcription factors, small RNA (sRNA) post-transcriptional regula­
tors have been shown to play a critical role in biofilm formation
(Fernández et al., 2016; Pusic et al., 2016). Protein kinase C-mediated
Mitogen-Activated Protein (MAP) kinase pathways and protein phos­
phatases are vital. In Candida albicans, two main transcription factors,
Bcr1p and Zap1p, regulate the expression of adhesins and the production
of β-1,3 glucan during biofilm formation (Nobile et al., 2009; Srikantha
et al., 2013). During C. albicans infection transcription factor, Fks1p
regulates the level of ECM polysaccharide in the biofilm (Taff et al.,
2012). In Aspergillus fumigatus, a reduction in adhesion and changes in
cell surface and ECM production were observed in the absence of MAP
kinase activity, especially those due to SakA, MpkA, and MpkC. Phos­
phatase mutants (ΔptcB and ΔsitA), having low cellular integrity and
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Ecotoxicology and Environmental Safety 264 (2023) 115389
reduced phosphorylation of MpkA and SakA, have been used to
demonstrate their role in inducing these changes (Manfiolli et al., 2018).
Transcriptomic studies have also supported the role of sRNA in
promoting gene expression in the ECM and curli fimbriae in
P. aeruginosa (Gómez-Lozano et al., 2012). Among over 200 sRNAs re­
ported in P. aeruginosa genomes, SrbA was proposed to play a role in
biofilm formation and pathogenicity. srbA deletion mutants had 66%
lower biofilm mass, and their infection resulted in a mortality rate of
39% over 72 h in Caenorhabditus elegans compared to 78% in the control
(Taylor et al., 2017). In P. aeruginosa, the sRNA ErsA contributes to the
regulatory system involved in its pathogenicity factors, including
motility and biofilm formation. Studies using genetically modified
P. aeruginosa strains have shown that ErsA influences EPS production
and positively regulates biofilm formation and maturation processes
(Ferrara et al., 2015; Falcone et al., 2018). It acts through the
post-transcriptional modulation of the transcription factor AmrZ by
targeting the algC gene encoding virulence factors (Ferrara et al., 2020).
In P. aeruginosa, multiple regulatory systems contribute to their patho­
genicity. A few hundred virulence factors are QS-mediated. The role of
sRNA (Qrrs) in regulating QS-mediated virulence factors has provided
significant insights into developing antimicrobial drugs. Although
transcriptome sequencing has identified potential intergenic sRNAs,
only the roles of a few have been well-defined. The sRNAs PrrH, PhrS,
PrrF, and AmiL are involved in expressing virulence factors, motility,
and biofilm formation (Thi Bach Nguyen et al., 2018; Lu et al., 2019; Pu
et al., 2022).
2.3. Quroum sensing
Bacteria express specific characteristics that are only beneficial if
they are produced in "large" quantities. These genes are expressed in a
synchronized manner when bacterial population density is high
(Papenfort and Bassler, 2016; Kalia et al., 2019). The expression of
QS-based genes leads to biofilm formation, sporulation, and antibiotic
biosynthesis. The QSS is involved in the biosynthesis of signaling mol­
ecules released into the milieu and sensed by specific receptors. The
signal-receptor complex is translocated into the cell and initiates tran­
scription upon binding to gene promoters. This QS-mediated gene
expression begins bacterial biofilm formation by synthesizing auto­
inducer (AI) signal molecules (i) acylhomoserine lactones (AHLs), (ii) AI
peptides, and (iii) AI-2 (Papenfort and Bassler, 2016; Dong et al., 2021).
In addition, biomolecules with signaling characteristics include (i) fatty
acids, (ii) quinolones and diketopiperazines (PQS), (iii) ketones, and (iv)
epinephrine and norepinephrine (Koul et al., 2016; Zhou et al., 2017).
Biofilm-forming QSSs vary among bacterial species; AI-2 regulates ica,
bhp, and icaR in Staphylococcus epidermidis, and las, rhl, PQS, and inte­
grated QS in P. aeruginosa (Papenfort and Bassler, 2016; Lee and Yoon,
2017). In contrast to a single QSS, a small number of bacteria possess
multiple QSSs, including Bacillus, Clostridium, Pseudomonas, Sino­
rhizobium, Streptococcus, and Vibrio spp. These QSSs operate as hierar­
chical systems or networks (Koul et al., 2016).
Bacterial cyclic dimeric guanosine monophosphate (c-di-GMP) is a
signaling molecule. It mediates the production of the bacterial ECM,
leading to biofilm formation. The binding of C-di-GMP to membrane
proteins LapD and LapA and effector YcgR regulates the transition from
the planktonic to biofilm mode of lifestyle (López-Baena et al., 2019).
Norspermidine, a polyamine secreted by Vibrio cholerae, regulates bio­
film formation. The NspS/MbaA signaling system binds NspS protein to
norspermidine, inhibiting MbaA phosphodiesterase activity. Further­
more, it also enhances c-di-GMP concentration to promote biofilm for­
mation. Norspermidine within biofilm also assists in acquiring iron,
which helps strengthen biofilms (Wotanis et al., 2017). Levels of cAMP
increase from low in planktonic cells to high after a few rounds of
bacterial attachment/detachment. As this process progresses, irrevers­
ibly attached cells transform into strong biofilm structures (Armbruster
and Parsek, 2018).
V.C. Kalia et al.
Other molecules that regulate the transition of planktonic bacteria to
the biofilm stage are sRNAs. Environmental stress induces sRNA pro­
duction. This system promotes gene expression in the ECM and curli
fimbriae and represses flagellar genes (Taylor et al., 2017). Microbial
adhesion and ECM synthesis within biofilms are mediated by a signal
transduction system, in which kinases (SakA, MpkA, and MpkC) are
highly active. Phosphatase mutants (ΔptcB and ΔsitA), which have low
cellular integrity and reduced phosphorylation of MpkA and SakA, have
been used to demonstrate their role in inducing these changes (Manfiolli
et al., 2018).
2.4. eDNA
Bacterial cell lysis releases genomic DNA (gDNA) into biofilms. The
gDNA may or may not undergo structural changes (Turnbull et al., 2016;
Deng et al., 2020). A comparison between strong and weak
biofilm-forming strains of S. epidermidis showed that the former pro­
duced more eDNA (Montanaro et al., 2011). This observation was
further supported by a comparison S. epidermidis and S. aureus, which
showed that higher biofilm formation in the former was due to the
mechanism of eDNA release via autolysin protein (AtlE) and cell lysis
(Dusane, 2017; Zatorska et al., 2017). In gram-negative bacteria, such as
Burkholderia pseudomallei, the quantity of eDNA in the biofilm is posi­
tively correlated with biomass thickness, independent of the ratio of
living and dead bacteria (Pakkulnan et al., 2019). The production and
release of eDNA follow different mechanisms; however, they generally
occur through cell lysis (Ibáñez de Aldecoa et al., 2017) or via mem­
brane vesicles (Toyofuku et al., 2019). QS-mediated release of eDNA
also occurs through 4–hydroxy-2–alkylquinolines (HAQs). In
P. aeruginosa, HAQs produce reactive oxygen species (ROS) that damage
the cell membrane and release eDNA (Tahrioui et al., 2019). Abundant
biofilms then form around the fragmented eDNA. The addition of DNase
I-digested gDNA to P. aeruginosa enhances its biofilm-forming ability
(Deng et al., 2020).
eDNA contributes to biofilm fitness through different mechanisms. It
regulates phenazine retention, redox recycling, and extracellular elec­
tron transfer to the bacterial populations within biofilms (Saunders
et al., 2020). It also promotes antibiotic resistance in bacteria within
biofilms by inactivating cationic antibiotics through HGT (Keeling and
Palmer, 2008; Saxena et al., 2019). eDNA interacts with various biofilm
components, specifically with a high affinity or non-specifically with a
low affinity. eDNA binds to various virulence factors and ribosomal
proteins to promote biofilm formation (Graf et al., 2019). eDNA-protein
interactions stabilize anionic eDNA and facilitate bacterial aggregation
(Arenas and Tommassen, 2017; Kavanaugh et al., 2019). Complexes of
host factors and histone-like proteins of the DNABII protein family bind
to DNA with high affinity in the extracellular matrix of biofilms (Devaraj
et al., 2018). Interactions among eDNA, EPS, and PsI in P. aeruginosa
promote bacterial adhesion and growth (Wang et al., 2015). In addition,
eDNA interacts with phenazine, which participates in cell signalling,
biofilm formation, iron acquisition, and antibiotic resistance (Schiessl
et al., 2019; Zhu et al., 2019). In P. aeruginosa, phenazine enhances
electron transfer, leading to stronger binding of eDNA to the cell surface
and higher viscosity of the DNA and sputum (Das et al., 2013, 2015). The
more virulent behavior of pathogenic bacteria and their higher stress
resistance are attributed to divalent cation sequestration by eDNA
(Mulcahy et al., 2008; Johnson et al., 2013; Lin et al., 2018). Although
marked biotechnological advances have been made to overcome the
biological factors that lead to infectious diseases, the challenges of eDNA
and its interaction with various biofilm components are still difficult to
circumvent (Panlilio and Rice, 2021).
3. Antibiofilm strategies
In view of the rapid emergence of antibiotic resistance and its
persistence, there has been a shift in the focus of drug development from
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Ecotoxicology and Environmental Safety 264 (2023) 115389
killing bacterial pathogens to regulating pathogenicity and virulence
factors (Subhadra et al., 2018; Kalia et al., 2019). Antibiofilm strategies
have been targeting either the formation stages or the pre­
formed/mature biofilm through the disintegration of the preformed
biofilms, inhibiting QS, and non-QS regulatory mechanisms. Bacterial
surface, membrane, and secretory molecules contribute to their ability
to evade the host immune system and adhere to host cells. A preventive
measure to hind infections is to target bacterial adhesion, the initial step
in biofilm formation (Sharma et al., 2017; Dufrêne and Viljoen, 2020).
Anti-adhesion strategy has the latent advantage of not hindering bac­
terial growth resulting in minimal selective pressure (Parrino et al.,
2018). In subsequent stages of infection, mature biofilms can be
removed by degrading their matrix components. A few enzymes, such as
(i) deoxyribonucleases (DNases), (ii) polysaccharide-degraders, and (iii)
proteases, can effectively metabolize the various components of the
biofilm, leading to its disintegration. Mature biofilms are more difficult
to eradicate as these are more tolerant to antimicrobial agents due to an
alteration in the growth rate of bacterial cells embedded in the biofilm
matrix, horizontal transmission of genes conferring resistance, and the
emergence of resistance mutants (Ito et al., 2009; Savage et al., 2013). In
addition to biological techniques, some studies have used electro­
chemical and photodynamic approaches to eradicate biofilms (Sultana
et al., 2016; Darmani et al., 2018; Gholibegloo et al., 2018; Srinivasan
et al., 2021).
3.1. Anti-Adhesion
The adhesion of bacterial cells to the surfaces involves various fac­
tors, such as surface proteins, pili, and exopolysaccharides (Asadi et al.,
2019). The most prevalent antibiofilm approaches involve biocides,
antibiotics, and ion coatings. The non-specific adhesion of E. coli can be
inhibited by the herbicide 2,4-dichlorophenoxyacetic acid (Bhat et al.,
2018). Coating medical devices, such as catheters, with antibiotics, such
as rifampin, minocycline, gentamicin, and norfloxacin, could signifi­
cantly reduce S. aureus and UTIs (Ramos et al., 2011). Telithromycin, a
semi-synthetic antibiotic, is effective against biofilms formed by
S. aureus, E. faecalis, and E. faecium strains. A 35–40% reduction has
been reported against biofilm formation and for treating established
biofilms. The effect of telithromycin could be enhanced from 40% to
70% in combination with ampicillin (Xiong et al., 2021). Antimicrobial
peptides, such as peptide 1018 and lantibiotics, have been found to
interfere with the biofilm formation process in several pathogens,
including P. aeruginosa, E. coli, Acinetobacter baumannii, K. pneumoniae,
S. aureus, Salmonella Typhimurium, and Burkholderia cenocepacia (De la
Fuente-Núñez et al., 2014), as well as S. aureus, and S. epidermidis
(Saising et al., 2012). A peptide derived from β-neurexin could block
surface attachment and adhesion by bacteria, leading to the complete
inhibition of biofilm accumulation (Feuillie et al., 2017). Immobilizing
antimicrobial agents, such as N-alkylpyridinium bromide, to poly
(4-vinyl-N-hexylpyridine) resulted in a 99% inhibition of infections on
medical devices caused by S. epidermidis, E. coli, and P. aeruginosa.
Exposure to subinhibitory concentrations of sodium hypochloride in­
duces structural changes in Listeria monocytogenes biofilms and strongly
reduces the ability of cells to adhere to polystyrene surfaces. Inciden­
tally, it also represses the expression of genes responsible for motility
and QS signaling activity (Bansal et al., 2021).
Thymol (a phenol derivative) treatment (100 μg/mL) was effective
in reducing MRSA adhesion on various surfaces coated with human
plasma. It also inhibited biofilm formation by 88%. This treatment is
mediated by the repression of sarA, which reduces the production of
adhesins and hemolysins (Valliammai et al., 2020). A unique feature of
polyphenols extracted from fruits is their ability to inhibit biofilm for­
mation and destroy preformed biofilms inhabited by Rothia dentocariosa
and other Streptococcus spp., such as S. mutans, S. oralis, and S. mitis. A
reduction in the adhesion of bacteria to surfaces results in the inhibition
of biofilm formation by up to 97.3%. In addition, disruption of
V.C. Kalia et al.
preformed biofilms by up to 80% has been reported after treatment with
various combinations of phytocompounds (Sateriale et al., 2020).
Poloxamer 338, with surfactant properties, has been proposed as an
anti-biofilm agent. It targets catheter-associated urinary tract infections
by repressing the adhesion of pathogenic bacteria. A P388-coating of
35 nm on catheters could reduce 0.51–0.83 log10 of E. coli strains
Ec5FSL and Ec9FSL (Stirpe et al., 2020). Biosurfactants have been shown
to exert anti-adhesive activity against bacterial pathogens. The bio­
surfactant produced by the probiotics Lactobacillus acidophilus and
L. rhamnosus inhibits S. mutans biofilm formation by downregulating the
expression of gtfB/C and ftfL (Satpute et al., 2019; Tahmourespour et al.,
2019).
Screening studies identified monoclonal antibodies as anti-adhesive
agents that prevent interactions involving S. aureus I adhesin, Cn, and
laminin (Herman-Bausier et al., 2016). The adhesion of bacterial cells to
surfaces, such as the surface protein SdrC of S. aureus, is a critical
component of biofilm formation (Wang et al., 2021). Another technique
for inhibiting bacterial adhesion involves blocking
carbohydrate-binding adhesins. Chemically-modified Galα1–4 Gal de­
rivatives inhibit streptococcal adhesin SadP (Haataja et al., 2018).
Coating titanium with DNase I inhibits bacterial adhesion and
biofilm-forming abilities (Ye et al., 2017). Chelators reduce the avail­
ability of metal ions in the biofilm matrix. Silver ions (AgNPs), along
with antibiotics, show enhanced antibacterial activity by interfering
with the DNA replication, protein synthesis, and respiratory process of
E. coli, S. aureus, Klebsiella species, P. aeruginosa, S. typhimurium, Candida
albicans (Secinti et al., 2011; Chernousova and Epple, 2013; Besinis
et al., 2017). Despite the demonstration of various anti-adhesive agents,
their application in treating infections caused by pathogens at the clin­
ical level is still lacking. These can be recommended as supplementary
approaches (Vuotto and Donelli, 2019).
3.2. Anti-extracellular matrix components
An approach to degrade biofilm targets its major components: exo­
polysaccharides (EPS), extracellular DNAs (eDNAs), and proteins.
(Flemming and Wingender, 2010; Mann and Wozniak, 2012). EPS and
eDNA are the major factors that regulate and promote antibiotic resis­
tance by delaying the diffusion of antimicrobials or promoting the
transmission of antibiotic-resistance genes (Mulcahy et al., 2008). The
disintegration of the biofilm exposes the sessile cells, making them
susceptible to host immune defense, and much lower concentrations of
antibiotics than those required against their free-living counterparts
(Kaplan, 2009).
eDNA, an integral element of the extracellular polymeric matrix
(EPM). Deoxyribonuclease I digests eDNA present in the biofilms of a
wide range of bacterial pathogens: Staphylococcus strains, A. baumannii,
E. coli, Haemophilus influenzae, K. pneumoniae, Psuedomonas aeruginosa,
etc. (Kaplan et al., 2012). Campylobacter jejuni strain RM1221 is a poor
biofilm former due to the presence of genes encoding eDNase. eDNase
from strain RM1221 could efficiently disrupt C. jejuni NCTC 11168
biofilms formed on steel surfaces. A surface treatment with eDNase can
disrupt even the preformed biofilms (Brown et al., 2015).
DNases have generally been observed to prevent biofilm formation
rather than degrade pre-formed biofilms (Okshevsky et al., 2015).
DNAse treatment has been reported to be effective during the early
stages of biofilm formation (< 6 h old). Dornase alfa (engineered human
DNAse I) is used to treat patients with cystic fibrosis (CF). The degra­
dation of eDNA reduces spitum viscosity (Wagener and Kupfer, 2012).
DNase breaks down the phosphodiester linkages of eDNA, leading to the
inhibition of biofilm formation in Gram-positive and Gram-negative
bacteria. Treatment leads to changes in various characteristics of the
bacterial community, including biomass, morphology, and architecture.
Treatment with DNase I-like 2 (DNase IL2) (present in human skin)
efficiently suppresses biofilm formation by P. aeruginosa and S, aureus,
which demonstrates the role of eDNA in biofilm development (Eckhart
5
Ecotoxicology and Environmental Safety 264 (2023) 115389
et al., 2007). DNase 1 (5.0 μg/mL) caused a 40% decrease in biofilm
biomass. DNase 1 in combination with β-lactams resulted in an addi­
tional 10- to 15-fold decrease in viable bacterial counts. DNase 1 plus
azithromycin decreased cell viability in biofilms by 2- to 4- fold in
Gram-positive bacteria (S. aureus, and Streptococcus pyogenes) and by
20-fold in Haemophilus influenzae (Tetz et al., 2009). DNase (NucB) ob­
tained from Bacillus licheniformis disperses eDNA present in the EPS
matrix of biofilms formed by Gram-negative and Gram-positive bacteria,
which demonstrates that bacteria can exploit these enzymes to disperse
biofilms formed by their competitors (Nijland et al., 2010). The inhibi­
tory effects of the extracellular protease and DNase from Bdellovibrio
bacteriovorus HD100 on S. aureus biofilms have been demonstrated. This
has been proposed as an approach for preventing the invasion of
S. aureus into human epithelial cells (Monnappa et al., 2014).
Treating the biofilms during the early development stage with DNase
I (0.1 U/100 μl) significantly inhibited growth and effectively disrupted
(>85%) 72-h-old mature C. jejuni biofilms (Kim et al., 2017)(Table 2). A
combination of DNaseI-benzalkonium chloride (BAC) caused a 3–5 log
CFU/mL disruption in a 48-h-old dual (E. coli + L. monocytogenes)
pathogenic species biofilm on a stainless-steel surface.
Cellulase/pronase-BAC combination was less effective than DnaseI. The
efficiency of DnaseI-BAC was also effective in Pseudomonas fluorescens –
L. monocytogenes mixed culture biofilms. For effective biofilm removal
with cellulase, a concentration of 1000 µg/mL was required (Rodrí­
guez-López et al., 2017). Taking advantage of the competitive behavior
of two types of foodborne pathogens, the supernatants of the
non-biofilm formers degraded C. jejuni NCTC11168 biofilm owing to a
16% reduction in eDNA. This approach could eradicate the pre-existing
biofilm entirely (Jung et al., 2017). DNase I has been successfully used to
target eDNA to assist the action of antibiotics in eliminating CF-related
P. aeruginosa biofilms. A combination therapy (GSH + DNase I + cip­
rofloxacin 5 µg/mL) could significantly reduce biofilm viability by
13–35%. An additional benefit of this combined treatment is the GSH
based assistance in the re-growth of the damaged tissue because of the
recovery of the host cells (Das et al., 2017).
Pre-treatment of P. aeruginosa PAO1 in the presence of 5 µg/mL
DNase caused a 68.6% reduction in biofilm formation over a period of
24 h. In contrast, biofilms treated with 10 µg/mL DNase exhibited a 70%
reduction. Here, the addition of Mg2+ ions (10 mM) enhanced the effi­
ciency of post-treatment DNase activity (5 µg/mL). It resulted in a 90%
reduction in the preformed biofilm within 5 min of contact time. How­
ever, DNase treatment was less effective against mixed-species biofilms
formed by P. aeruginosa PAO1, Klebsiella spp., E. faecalis, and
S. Typhimurium, and S. aureus (Sharma and Pagedar Singh, 2018). Under
in vitro conditions, biofilm treatment with DNase1 (0.001 or
0.002 mg/mL) significantly affected (reduced) the growth (log10 CFU)
Streptococcus mutans UA 159 (OMZ 918), and S. oralis SK 248 (OMZ 607)
induced biofilm. However, proteinase K treatment alone or in combi­
nation with DNase I showed an enhanced growth of S. mutans UA 159
(OMZ 918), and S. oralis SK 248 (OMZ 607) induced biofilms. Confocal
images of biofilms treated with a combination of DNaseI and proteinase
K resulted in less dense biofilms. Complete degradation of eDNA within
biofilms treated with DNase I was also recorded with Actinomyces oris
OMZ 745, Candida albicans OMZ 110, and Fusobacterium nucleatum
KP-F2 (OMZ 596) (Karygianni et al., 2020b). Various studies have been
conducted using commercially available DNases, such as DNase I
derived from bovine pancreas, DNase 1L2 from human keratinocytes,
Dornase alfa (recombinant human DNase), λ exonuclease (viral DNase),
NucB, and streptodornase produced by Bacillus licheniformis and Strep­
tococcus spp., to disperse pathogenic bacterial biofilms (Fleming and
Rumbaugh, 2017). It may be concluded that a combination therapy of
DNase I and antimicrobials can be more effective than either alone.
Although eDNA is a critical target for inhibiting biofilm formation,
its interactions with other molecules can also be exploited as targets for
anti-biofilm drugs. DNase treatment increases biofilm permeability.
Monoclonal antibodies (MAbs) specifically targeting DNABII disrupt
V.C. Kalia et al. Ecotoxicology and Environmental Safety 264 (2023) 115389

Table 1
Bioactive molecules inhibiting quorum sensing mediated bacterial biofilms.
Bioactive molecule Pathogen QS inhibition Surface Biofilm inhibition Effective Applications References
(Source) (I); concentration (EC);
Removal of Applied
preformed (R) (%) concentration (AC)
of inhibitor

Phytochemicals Pseudomonas AHL PS MTP I: 11.82–84.37; R: MEKB: methanolic For treating Sarkar et al. (2015)
(Kalanchoe aeruginosa biosynthesis 46.8–91.7 (viable extract of K. pathogenic infections
blossfeldiana) cells) blossfeldiana in humans
EC: 30, and 62.5 μg/
mL;
AC: 15, 30, 62.5,
125 μg/mL; (MIC:
250 μg/mL)
Curcumin (280 µg/ Aeromonas sobria AHL PS MTP I: 93.95%; Curcumin liposomes Treating opportunistic Ding et al. (2017)
mL) biosynthesis R: NR EC: 280 µg/mL; food pathogens
AC: 280–420 µg/mL
AHL lactonase Acinetobacter AHL Glass I: 80% with Aii20J; EC: Aii20J at 20 μg/ Prevention and Mayer et al. (2020)
(Aii20JB) and baumannii ATCC® biosynthesis coverslips 20% with DNase; mL; treatment of bacterial
DNase 17978™ R: NR AC: Aii20J at 20 μg/ infections such as
mL, DNase at 2 U/ pneumonia,
mL; α-amylase septicemia, skin,
(10 U/mL) (alone or wound, and urinary
in combinations) tract infections
Spathulenol P. aeruginosa AHL PS: MTP I: 80% by plant EC: 10 μl of 0.1 mg/ Potential use as an Luciardi et al. (2016)
(Merremia biosynthesis extract); mL extracts effective
dissecta) R:NR AC: 0.1 mg/mL antipathogenic drug
extracts in
DMSO–H2O (50:50)
Lactonase (SsoPox-I P. aeruginosa AHL MTP Variable; - SsoPox-I: Therapeutic usage Hraiech et al. (2014)
protein) degradation EC: 1 mg/mL against pneumonia
(250 μl) (Rat
model);
AC:0.05–50 µg
(100 μl)
Lactonase (Bacillus P. aeruginosa PAO1 AHL MTP 60; - EC: Cell extracts Suppression of Rajesh and Ravishankar
and Enterobacter) and PAO1-JP2. degradation 100 μg/mL virulence in humans Rai (2014)
AC: Cell extracts and plants
100 μg/mL
Lactonase Acinetobacter AHL Medium 68.75%; 2 µm3/ EC: AHL-lactonases, Effective Chow et al. (2014)
(Geobacillus baumannii S1 degradation plate μm2 10 μl of purified antipathogens against
kaustophilus) enzyme (40 mg/ human bacterial
mL); pathogens
AC: AHL-lactonases,
10–30 μl of purified
enzyme (40 mg/mL)
Fatty acids P. aeruginosa PA01 AHL PS 48.29–53.99; - EC: Not defined Promising medicinal Pejin et al. (2014)
(Ochridaspongia degradation AC: 0.5 MIC; MIC properties
rotunda) was 30 mg/
mL O. rotunda
samples
Hexasaccharide Staphylococcus AHL PS 100; 71.5 EC: 5 mg/mL CHS Prevent bacterial Srivastava et al. (2015)
(Equus caballus) aureus degradation AC: 2–10 mg/mL for pathogenicity
MIC
AHL lactonase P. aeruginosa AHL Medium 60;- EC: 20 μl of purified Potential use as El Aichar et al. (2022)
(Bacillus degradation plate cell extracts (PCE) probiotics and
thuringiensis) AC: 20 μl PCE of the antibiofilm agent
Bacillus strains
dilutions from 10− 1
to 10− 5
AHL lactonase Streptococcus AHL Medium 63;- EC: 20 μl of purified Potential use as
(Bacillus subtilis) mutans degradation plate cell extracts (PCE) probiotics and
AC: 20 μl PCE of the antibiofilm agent
Bacillus strains
dilutions from 10− 1
to 10− 5
Diketopiperazines Lelliottia amnigena AHL Medium 70–80;- EC: 100 μl of crude Reduce pathogenicity. Kachhadia et al. (2022)
(by Bacillus degradation plate extract Reduction in the
cereus) AC: 100 μl of crude emergence of
extract antibiotic-resistant
bacteria
Passiflora edulis Chromobacterium AHL Medium 51–90;- EC: 2 mg/mL Reduced biofilm Venkatramanan et al.
violaceum degradation plate (P. edulis extract) formation (2020)
AC: 0.5, 1.0, and
2.0 mg/mL
(continued on next page)

6
V.C. Kalia et al. Ecotoxicology and Environmental Safety 264 (2023) 115389

Table 1 (continued )
Bioactive molecule Pathogen QS inhibition Surface Biofilm inhibition Effective Applications References
(Source) (I); concentration (EC);
Removal of Applied
preformed (R) (%) concentration (AC)
of inhibitor

Coumarin Porphyromonas Autoinducer-2 MTP -;40.4 EC: 200 μM Treatment of He et al. (2022)
gingivalis inhibition AC: 25–200 μM periodontitis
(periodontal
multispecies biofilm)
Isothiocyanates P. aeruginosa Block signal MTP NR;- EC: 200 μM Treating opportunistic Ganin et al. (2013)
(Broccoli) receptor (Sulforaphane) bacterial pathogens.
AC:50–200 μM
(Sulforaphane)
Phenolic compound P. aeruginosa Block signal MTP Variable;- EC: 1.36 mM Suppressing virulence Gutiérrez-Barranquero
(Coumarin) receptor Coumarin and broad spectrum et al. (2015)
AC: 10.0, 5.0, 2.0, drug
1.71, 1.36 and
1.0 mM Coumarin
Polyphenols from P. aeruginosa Block signal MTP >90;- EC: 4.5 μg/mL Affect Quorum- Prateeksha et al. (2017)
honey (HP receptor SeNPs@HP sensing mediated
AC: SeNPs: 7.5 and pathogenicity; Anti-
25 μg/mL; virulence activities
HP: 0.6% and 5%; (in-vitro and in-vivo)
SeNPs@HP: 4.5 and
25 μg/mL
Hamamelitannin S. aureus Inhibits gene Artificial 90.9% (in CFUs/ EC: HAM (120 μg/ Wound healing Brackman et al. (2016)
+ antibiotic transcription dermis biofilm, mL), VAN (250 μg/
(Vancomycin) P. aeruginosa); 70% mL);
(in CFUs/ biofilm in AC: HAM
S. aureus Mu50); NS (0.5–500 μg/mL)
and vancomycin
(1–500 μg/mL)
Dioon spinulosum Porphyromonas Down- MTP 77.1; - EC: Not defined Application in treating Elekhnawy et al. (2022)
extract gingivalis regulation of AC: 125–500 μg/mL wounds, respiratory
genes for QS Dyer Ex Eichler and urinary tract
and biofilm extract infections

‘-‘ Not available; AHL: Acylhomoserine lactone; PS: Polystyrene; MTP: Microtiterplate.

preformed biofilms (Novotny et al., 2016). In contrast, proteinase K
treatment did not cause any additional changes in permeability (Ran­
drianjatovo-Gbalou et al., 2017). It has been suggested that targeting
DNABII is more effective because it is an integral part of the vertices of
DNA strands (Novotny et al., 2016). DNase I and alginate lyase disin­
tegrate the biofilms formed by Enterococcus faecalis and E. faecium in the
urinary tract. These enzymatic treatments enable vancomycin to
significantly reduce biofilm hydrophobicity and structure, and cell
viability (Torelli et al., 2017).
The major ECM degrading enzymes are hydrolases, proteases, and
nucleases (Li and Lee, 2017). Dispersin B (glycoside hydrolase) hydro­
lyzes poly-N-acetylglucosamine (PNAG), leading to a marked reduction
in the infections caused by Staphylococcus spp. and E. coli. A combination
of Dispersin B and triclosan reduced biofilm forming ability of S. aureus,
S. epidermidis, and E. coli (Darouiche et al., 2009). Proteinase K cleaves
the protein component of the biofilm leading to its dispersal. Dispersin B
treatment followed by Proteinase K or trypsin was more effective in
eradicating Staphylococcus biofilms. The major limitation of this
approach is the allergic reactions elicited by the host (Chen et al., 2013).
The quantity of EPS present in the ECM regulates the penetration and
diffusion of antimicrobial compounds into Salmonella enterica Typhi­
murium ATCC14028 biofilms. Salmonella biofilms produced by an
isogenic ΔcgsD mutant had significantly reduced EPS production, which
reduced cell adhesion and 72% lower biofilm biomass. EPS inhibitor
(2-cyclopentenyl-5-(4-chlorophenyl)− 2-aminoimidazole dissolved in
DMSO) reduced biofilm cells in the wild-type strain from 2.3 C to
1.8 CFU x 109, whereas in the sensitive strain, the reduction was from
1.8 to 1.2 CFU x 109. The impact on biofilm biomass (OD 570) WT was
0.28 and 0.1; in contrast, in the sensitive strain, it got reduced from 0.24
to 0.02. The sensitivity to ciprofloxacin and hydrogen peroxide (H2O2)
on monoculture biofilms of wild-type strain ATCC14028 and isogenic
ΔcsgD mutant (as survival %) were 17% and 2%, respectively, with
antibiotic and 50% and 10% respectively with disinfectant (Dieltjens
et al., 2020) (Table 2). Preformed biofilms can be destabilized and dis­
rupted by EPS metabolism. The action of hydrolytic enzymes on EPS
components can significantly alter biofilm integrity (Kalia et al., 2018).
The role of proteases in biofilm dispersal has been demonstrated for
many pathogens: (i) Aureolysin and V8 serine protease (SspA) act against
S. aureus biofilms by degrading Bap and clumping factor b (Gustafsson and
Oscarsson, 2008), (ii) Trypsin disperses P. aeruginosa biofilms (Banar et al.,
2016). Thymol in thyme oil (0.5 mg/mL), and Proteinase K (50 and
100 µg/mL) combinations effectively inhibited adhesion and proliferation
of E. coli O157:H7. On the other hand, (50–100 µg/mL PK +
0.25/0.5 mg/mL TO) had synergistic biofilm-dispersal effects at 24 h (Cui
et al., 2016). A few other examples of the role of proteases are as follows:
(i) LapG Protease A triggers dispersal by modifying the EPS-binding pro­
tein LapA; (ii) proteinase K targets peptide bonds and disperses biofilms
produced by many bacteria; (iii) Spl protease A disperses S. aureus by
cleaving EbpS proteins associated with the cell wall; (iv) streptococcal
cysteine protease (SpeB) A hydrolyzes surface proteins M and F1; and (v)
protease surface-protein-releasing enzyme (SPRE) acts by releasing anti­
gen P1 (Fleming and Rumbaugh, 2017).
A crude extract of Adiantum philippense L. decreased the EPS content
of biofilms. It attacks adhesins and disrupts preformed biofilms. At MIC the
inhibition (%) of adhesion and of preformed biofilms by A. philippense was
quite varied, ranging from as low as 37.34 and 44.10 for Shigella flexneri,
50.26% and 56.54% for P. aeruginosa, 54.73 and 62.72 for E. coli, and
60.92% and 70.58% for S. aureus, respectively (Adnan et al., 2020). The
human systemic amyloid precursor transthyretin eradicates bacterial
biofilms by inhibiting CsgA amyloid-β aggregation. Curli amyloid fibers
along with cellulose are an integral component of EPM, in E. coli and
Bacillus subtilis biofilms. Engineered nontetramer-forming monomer
(M-TTR) was more efficient than wildtype-TTR tetramer (Jain et al.,
2017).

7
V.C. Kalia et al. Ecotoxicology and Environmental Safety 264 (2023) 115389

Table 2
Bioactive molecules inhibiting biofilm matrix components.
Bioactive molecule Bacterial pathogen Effective [Applied] Biofilm inhibition/ References
concentration of inhibitor removal (%)
Name Activity

DNase I Oxidative Campylobacter jejuni EC: 4 U/mL Complete Brown et al. (2015)
AC: 4 U/mL
DNase I Oxidative C. jejuni EC: 0.1 U/100 μl -a;> 85 Kim et al. (2017)
AC: 0.1, 0.01, and 0.001 U/100 μl
DNase I + benzalkonium Oxidative Escherichia coli EC: 400 µg/mL and 600 µg/mL 3–5 log CFU/mL reduction Rodríguez-López
chloride (BAC) and + Proteolytic + Listeria monocytogenes AC: et al. (2017)
Pronase-BAC Enzymes: 200, 400, 700, and
1000 µg/mL;
BAC (Benzalkonium chloride): 25, 50,
100, 250, and 500 µg/mL
BAC-PRN/DnaseI: 400 µg/mL
(enzyme)
Dnase I Oxidative Campylobacter jejuni and EC: 50 μl of ~108 CFU of the cell 16% reduction in eDNA; Significant Jung et al. (2017)
C. coli suspensions
AC: 50 μl of ~108 CFU of the cell
suspensions
DNase I + GSH Oxidative Pseudomonas aeruginosa EC: DNase I (40 U) or GSH (5 mM); Combination Therapy (CT) (GSH + Das et al. (2017)
+ Pyocyanin ciprofloxacin (5 µg/mL), DNase I + ciprofloxacin 5 µg/mL)]:
AC: DNase I (40 U) or GSH (5 mM); biofilm viability significantly reduced
ciprofloxacin (5, 10, and 15 µg/mL) to 13–35%.
DNase I + GSH Oxidative P. aeruginosa EC: DNase I (40 U) or GSH (5 mM); -;> 85
+ Ciprofloxacin ciprofloxacin (5 µg/mL),
AC: DNase I (40 U) or GSH (5 mM);
ciprofloxacin (5, 10, and 15 µg/mL)
DNase I Oxidative P. aeruginosa PAO1 EC: DNase I (5–10 µg/mL);, 68.6;- Sharma and
AC: DNase I (0–50 µg/mL) Pagedar Singh
DNase I + Mg2+ ions Oxidative P. aeruginosa PAO1 EC: DNase I (5 µg/mL);, -;90 (2018)
AC: DNase I (0–50 µg/mL)
DNase I Oxidative Streptococcus mutans UA EC: DNase I (0.001 mg/mL); 100 reduction in eDNA;- Karygianni et al.
159 (OMZ 918), and AC: DNase I (0.001–0.002 mg/mL) (2020b)
S. oralis SK 248 (OMZ
607)
2-cyclopentenyl-5-(4- EPS degrading Salmonella enterica EC: EPS inhibitor# - 50 μM Reduced EPS and cell adhesion. Dieltjens et al.
chlorophenyl)− 2- Typhimurium AC: EPS inhibitor - 50 μM Biofilm cell reduction inn resistant (2020)
aminoimidazole ATCC14028 strain 2.3–1.8 CFU x 109 where as in
the sensitive strain the reduction was
from 1.8 to 1.2 CFU x 109.
Aureolysin and V8 serine EPS degrading S. aureus EC: ND Degrading Bap and clumping factor Gustafsson and
protease (SspA) AC: ND Oscarsson (2008)
α-mannosidase, EPS degrading P. aeruginosa ATCC EC: mannosidases: 0.02 unit/mL and Degrading biofilms: 5–60% Banar et al. (2016)
β-mannosidase 27853 trypsin: 0.75 μg/mL Best: Trypsin
(Hydrolases) and AC: mannosidases: 0.005, 0.01, 0.015,
trypsin (Protease) 0.02 and 0.03 unit/mL and trypsin:
0.08, 0.175, 0.35, 0.75 and 1.5 μg/mL
Proteinase K + thyme oil Antibacterial E. coli O157:H7 EC: PK - 50 and 100 µg/mL; TO - 3.84–3.89 logs lower cells: Inhibit Cui et al. (2016)
(TO) 0.5 mg/mL) (Biofilm inhibition) adhesion and proliferation. Biofilm
EC: PK - 50 and 100 µg/mL; TO - 0.25/ dispersal (Not quantified)
0.5 mg/mL) (Biofilm dispersal).
AC: PK - 25, 50 and 100 µg/mL; TO -
4.0, 2.0, 1.0, 0.5 and 0.25 mg/mL)
Adiantum philippense L.+ EPS degrading Shigella flexneri, P. EC: 200 μl (MIC) + 100 μl (1000 μg/ 37–60.92; 44.10–70.58 Adnan et al. (2020)
chloramphenicol aeruginosa, E. coli, and S. mL)
aureus AC: 100–200 μl (plant extract -
1000 μg/mL) + chloramphenicol:
100 μl (1000 μg/mL)
Transthyretin (TTR) Anti-ECM E. coli and Bacillus EC: 1:1 CsgA: M-TTR 80;- Jain et al. (2017)
subtilis AC: CsgA (15 µM); 1:1 CsgA: M-TTR
and 50 µM M-TTR
Human monoclonal Anti-ECM Salmonella EC: 0.5 mg/mL 70 (Reduced staining); 40 Tursi et al. (2020)
antibody (mAb) 3H3 Typhimurium AC: 0.5 mg/mL
Vancomycin and Anti-ECM MSSA and MRSA EC: 10 mg/mL (Erdosteine) Reduced IC50 values Pani et al. (2022)
linezolid + erdosteine AC: 2, 5 and 10 mg/mL (Erdosteine)
alone or in combination with
Antibiotics (0, 0.5, 1, 5, 10, 20, 50,
100 and 200 times the MIC)
a
Not available; EPS: Exopolysaccharides; GSH: Reduced glutathione; IC50, half-maximal inhibitory concentration; MRSA: methicillin-resistant S. aureus; MSSA:
methicillin-sensitive S. aureus. #: 2-cyclopentenyl-5-(4-chlorophenyl)− 2-aminoimidazole dissolved in DMSO

The biofilm matrix formed by Salmonella spp. and E. coli contains vancomycin and linezolid against MSSA- and MRSA-induced biofilms
approximately 85% curli amyloid fibers. Human monoclonal antibody was further enhanced in the presence of erdosteine, a thiol-based drug,
(mAb) 3H3, which binds pan-amyloid epitopes, plays a dual role in over a period of 24 h. Erdosteine facilitated the penetration of the
preventing and eradicating biofilms (Tursi et al., 2020). The activity of antibiotic into the biofilm and disrupted EPS (Pani et al., 2022). Various

8
V.C. Kalia et al.
studies have revealed co-applicating these enzymes results in synergistic
degradation of ECM components.
A few other antibiofilm agents observed to act against pathogenic
bacterial infections are the free fatty acids. P. aeruginosa produces cis-2-
decenoic acid to inhibit preformed biofilms induced by E. coli,
K. pneumoniae, P. mirabilis, S. pyogenes, B. subtilis, S. aureus, and
C. albicans (Abdel-Mawgoud et al., 2010). Xanthomonas campestris pro­
duce cis-11-methyl-2-decanoic acid, which can induce biofilm dispersal
by regulating the production of EPS-degrading enzyme (Deng et al.,
2014). However, fatty acids promote biofilm formation in B. subtilis
during the initial stages (Pedrido et al., 2013). Although it has been
observed that L-amino acids promote biofilm formation in P. aeruginosa,
however, D- and L- forms of tryptophan have the dual abilities of
inhibiting biofilm formation and it dispersal (Brandenburg et al., 2013).
Induction of biofilm dispersal in P. aeruginosa has been reported using
nitric oxide (NO), which can be generated from various compounds such
as sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO) and
S-nitroso-N-acetylpenicillamine (SNAP). A synergistic effect of NO was
reported along with antibiotics leading to dispersal of P. aeruginosa
biofilms formed in CF sputums (Howlin et al., 2017).
3.3. Anti-QS-mediated biofilm formation
Bioactive molecules produced by prokaryotes and eukaryotes can
potentially prevent QS-mediated biofilm formation (Kalia, 2013;
Gómez-Gómez et al., 2019). These QS inhibitors (QSIs) may target QSSs
at various stages, including signal biosynthesis, degradation of signal
molecules, competition for signals binding to cognate receptors, and
repression of QS-mediated gene transcription. As QSIs act below the
minimum inhibitory concentration (MIC) and do not restrict microbial
growth, we anticipate significantly reduced drug-resistant pathogens’
emergence rates (Koul et al., 2016; Koul and Kalia, 2017).
3.3.1. Affecting signal biosynthesis
Several QSIs have been shown to inhibit AHL biosynthesis, and
consequently, QS-mediated biofilm formation (Table 1). Kalanchoe
blossfeldiana leaf extract suppresses P. aeruginosa pathogenicity by
interfering with AHL biosynthesis. This results in 11.8–84.37% inhibi­
tion of biofilm formation (Sarkar et al., 2015). The use of Merremia
dissecta creeper extract, a medicinal plant, interferes with AHL biosyn­
thesis by 63–75%, and the biofilm-forming ability of P. aeruginosa ATCC
27853 by 55%. Spathulenol present in M. dissecta inhibits the
biofilm-forming potential by up to 80% in P. aeruginosa and by 71% in
S. aureus (Luciardi et al., 2016). Curcumin (2.5 μl of 280 µg/mL),
entrapped in liposomes, interacts with the LuxI-type protein and inhibits
the production of AHL. The production of C6-HSL, C10-HSL, and
C12-HSL was completely inhibited with curcumin liposomes, whereas
C4-HSL and C14-HSL were reduced up to 64.6% and 73.0% with free
curcumin, compared to 71.35% and 93.55% with curcumin liposomes,
respectively. As a result of the reduced AHL concentration, biofilm
formation was reduced by 93.35% in contrast to its control at 420 µg/mL
and free curcumin, which resulted in a reduction of only 51.9%. In
addition, curcumin reduces virulence factors’ expression in Aeromonas
sobria (Ding et al., 2017). In A. baumannii, QSS-abaI/R mediates motility
and biofilm formation. The purified QSI enzyme Aii20J reduced biofilm
formation in A. baumannii by 80% after 24 h. However, DNase signifi­
cantly reduced biofilm formation by 20%. A combined treatment
involving Aii20J and DNase did not lead to any improvement in the
results than those obtained with the QSI enzyme. In contrast, the
application of α-amylase did not affect biofilm formation. A strategy
involving induced mutations in AHL synthase AbaI and treatment with
the QSI enzyme Aii20J and DNase can significantly contribute to pre­
venting and treating A. baumannii infections (Mayer et al., 2020).
3.3.2. Inactivating signals
Degrading QS signals may block QS-mediated biofilm formation and
9
Ecotoxicology and Environmental Safety 264 (2023) 115389
subsequent pathogenicity. The enzymatic degradation of AHLs in­
activates QSSs. Lactonases and acylases produced by bacteria inactivate
AHLs by opening the lactone ring and splitting the acyl chain, respec­
tively (Kalia, 2013). Bacillus extracts, purified lactonases, and the het­
erologous expression of the aiiA gene can inhibit AHL-mediated QS
(Kumar et al., 2013; Noor et al., 2022). Biofilm formation by
P. aeruginosa in a rat model with highly lethal acute pneumonia was
observed to be reduced by lactonase (SsoPox-I) as a QSI. The reduction
was dose-dependent with a [C1/2]~170 µg/mL. The treatment led to a
55% reduction (from 75% to 20%) in the mortality rate (Table 1)
(Hraiech et al., 2014). Similarly, the lactonases of the endophytic bac­
teria Bacillus firmus PT18 and Enterobacter asburiae PT39 (extracts
100 μg/mL) inhibited biofilm formation by P. aeruginosa PAO1 and
PAO1-JP2. The inactivation of C4-HSL by the cell-free lysate of B. firmus
PT18 and E. asburiae PT39 inhibited biofilm formation of > 80% (Rajesh
and Ravishankar Rai, 2014). The wild-type GKL did not show any lac­
tonase activity against 3-OH-C12-HSL but could effectively hydrolyze
3-OH-C10-HSL. However, mutant GKL enzyme (E101G/R230C) could
inactivate 3-OH-C10-HSL and 3-OH-C12-HSL, which resulted in a
68.75% reduction in biofilm biomass (μm3/μm2) with respect to the
inactive mutant (Chow et al., 2014). The QSI present in crude extracts
from the freshwater sponge Ochridaspongia rotunda causes a 48.29%
(methanol extract) and 53.99% (acetone extract) inhibition of biofilm
formation by P. aeruginosa. The treatment also causes a 42–49%
reduction in pyocyanin production. The expression of QSI also affects
flagellar motility and twitching activity (Pejin et al., 2014). The hex­
asaccharide present in mare colostrum causes AHL degradation and
suppresses QS-mediated activities, such as biofilm formation and path­
ogenicity, by S. aureus. At 5 mg/mL, it curtails the antibiotic resistance
of methicillin-resistant S. aureus (MRSA) (Srivastava et al., 2015). The
irreversible hydrolysis of AHL by acylase PvdQ activity in a mouse
model demonstrated its potential as a therapeutic agent (Utari et al.,
2018). The AHL inhibitors 4-methoxycinnamic acid and 4-dimethylami­
nocinnamic acid have dual benefits, namely the inhibition of biofilm
formation and the enhancement of susceptibility to tobramycin (Cheng
et al., 2020).
Screening halotolerant strains of Bacillus revealed the presence of the
lactonase-encoding aii gene. The AHL degrading ability of Bacillus
thuringiensis DZ16 caused a 60% inhibition of biofilm formation by
P. aeruginosa. Lactonase of B. subtilis DZ17 reduced Streptococcus mutans
ATCC 25175 biofilm formation and biomass up to 63% in the xCELLi­
gence model (El Aichar et al., 2022). Prominent biomolecules produced
by Bacillus cereus RC1 were diketopiperazines, viz. cyclo(D-phenyl­
alanyl-L-prolyl), cyclo Phe-Val, cyclo(Pro-Ala), cyclo(L-prolyl-L-valine),
cyclo (Leu-Leu), and cyclo(-Leu-Pro). These biomolecules caused an
attenuation of 88.78%, 91.22%, and 97.59% of soft rot caused by Lel­
liottia amnigena RCE in cucumber, carrot, and potato, respectively. This
treatment reduced pathogenicity, especially biofilm formation, by
70–80% from 24 to 96 h after incubation without developing antibiotic
resistance (Kachhadia et al., 2022). Passiflora edulis extract containing
hexadecanoic acid and 2-hydroxy-1-(hydroxymethyl) Et ester in­
activates AHLs. Inhibition in EPS production by 35.9%, 52.2%, and
72.5% and reduction in biofilm formation by 51.8%, 69.4%, and 90.7%
were observed at the sub-MIC levels of 0.5, 1, and 2 mg/mL, respectively
(Venkatramanan et al., 2020). Periodontitis caused by biofilm-forming
Porphyromonas gingivalis is inhibited by coumarin. The downregulation
of biofilm-related gene expression was observed, which led to the in­
hibition of AI-2 activity. The antimicrobial effect of coumarin (200 μM)
against P. gingivalis was observed regarding reduced planktonic growth
and membrane permeability. The most effective reducing activities were
observed at 200 μM, affecting late-stage biofilm formation and dispersed
preformed biofilms (He et al., 2022).
3.3.3. Blocking receptors
Inhibition of QS-mediated gene expression can be achieved by
blocking receptors with analogs of signaling molecules.
V.C. Kalia et al.
Cinnamaldehyde and its derivatives exhibit vigorous inhibitory activity
against QS-mediated biofilm formation in P. aeruginosa and E. coli (Kalia
et al., 2019). Plant biomolecules (from broccoli), such as isothiocyanates
and glucosinolates, act as QSIs against E. coli and P. aeruginosa. Sulfo­
raphane inhibited the AHL receptor LasR binding at 50 µM, whereas
12 µM was sufficient to hinder biofilm formation by P. aeruginosa. In the
presence of either of the isothiocyanates (100 µM), namely sulforaphane
or erucin, a 70% reduction in QS-mediated pyocyanin synthesis in
P. aeruginosa was observed. The difference in the activities between the
two compounds seems to be due to differences in their hydrophobicity or
the slower oxidation of erucin (Ganin et al., 2013). Coumarin, a sec­
ondary metabolite present in cassia, bison grass, melilot, tonka bean
woodruff, and cinnamon, inhibits QS-mediated biofilm formation by
E. coli, Vibrio anguillarum, Edwardsiella tarda, and S. aureus. The phenolic
compounds of coumarin act as QSI by inhibiting AHLs, AI-2, and agr
QSS, which negatively affects rhlI- and pqsA-mediated gene expression
responsible for biofilm formation with other virulence factors in
P. aeruginosa (Gutiérrez-Barranquero et al., 2015). The Serratia marces­
cens biofilm-associated exopolysaccharides production, hydrophobicity,
and swarming motility were found to be inhibited by Piper betle extract.
At 500 µg/mL, the PBE inhibited biofilm formation and EPS production
by 65% and 43%, respectively, in S. marcescens. Bioactive compounds
act primarily by downregulating the expression of the QS genes fimA,
and fimC, which encode for fimbrial proteins responsible for adherence
and colonization (Srinivasan et al., 2016, 2017). Similar QS gene
downregulation due to QS receptor antagonists (tocopherols and phytol)
has also been reported in Diplocyclos palmatus leaf extract (Alexpandi
et al., 2019). The S. epidermidis biofilm inhibition efficacy of geraniol in
rats was shown through oral supplementation at a rate of 200 mg/kg
body weight for 3 days (Arunachalam et al., 2018). Selenium NPs,
intracellular drug delivery agents, and polyphenols present in honey
(HP) have shown superior QSI activity, targeting the LasR receptor. The
biofilm-forming ability of P. aeruginosa PAO1 was significantly reduced
by > 90%. Polyphenols of honey alone were ineffective at HP MIC 5%
and HP Sub-MIC 0.6% (Prateeksha et al., 2017). Phytochemicals, such as
garlic extract, act as QSIs by competitively blocking the binding of QS
signal molecules to transcriptional regulators such as LuxR and LasR in
P. aeruginosa (Mishra et al., 2020). Indole and its derivatives as QS signal
analogs exhibit a dose-dependent QSI effect, suppressing biofilm for­
mation, prodigiosin production, and motility at a MIC over a range of
2.5–5.0 mM. Indole derivatives cause 40–75% inhibition of biofilm
formation by S. marcescens (Sethupathy et al., 2020).
3.3.4. Inhibiting gene transcription
The targeting of gene expression related to biofilm formation and
pathogenicity by QSIs can help in treating infectious pathogens. Among
a wide range of biofilm inhibitors, cyclodextrins (CDs) (e.g., α- and
β-CDs) and their derivatives (methylated, quaternary amino, and poly­
mer) act as QSIs. Several studies have shown that QSI is induced by CD-
encapsulated QS signals, preventing binding to their cognate receptors.
It thus promotes the inhibition of QS-mediated pathogenicity, including
biofilm formation. Synthetic CD derivatives (6-alkylamino-α- or γ-CDs,
2-alkylamino-CDs, and 6,6’-dioctylamino-β-CD) act as inhibitors of QS-
mediated elastase production, a key regulator of antibiotic-resistant
biofilm produced by P. aeruginosa PAO1, violacein pigment production
of Chromobacterium violaceum, and the prodigiosin production of
S. marcescens (Morohoshi et al., 2013). Hydroxypropyl-β-CDs have been
shown to reduce the ability of P. aeruginosa outer-membrane vesicles,
which fuse with the lipid rafts of epithelial cells, to reduce CFTR Cl-
secretion. Hydroxypropyl-β-cyclodextrin (HPβCD) or methyl-­
β-cyclodextrin (MβCD) are equally effective in suppressing planktonic
growth and the biofilm-forming ability of P. aeruginosa (Barnaby et al.,
2019). α-CD and its methylated form can inhibit 90–98% of the
QS-mediated biofilm formation by P. aeruginosa. Changes in tempera­
ture were found to reduce the ability of α-CD at 0.1 mM from 78% at
30 ◦ C to non-significant levels at 22 ◦ C after 6 h (Berkl et al., 2022). In
10
Ecotoxicology and Environmental Safety 264 (2023) 115389
addition, α-CD (10 mM at 120 min) caused around 64% inhibition of
QS-mediated bioluminescence in Aliivibrio fischeri (Molnár et al., 2021).
In contrast to the studies mentioned above, others have shown the
potential of CDs in releasing antibiotics and QSIs in a sustained manner.
The QSI hamamelitannin (HAM), a nonpeptide analog, inhibits the RAP/
TRAP QS system in S. aureus, thereby increasing the susceptibility of
biofilms toward conventional antibiotics. This dual functionality of
HAM has several advantages. Wound care gauzes made of cellulose
functionalized with non-toxic hydroxypropyl-β-cyclodextrin were found
to release antibiotics (vancomycin) and QSI (HAM) in a sustained
manner, causing: (i) a variation from 20% to 55% in biofilm formation,
(ii) in the case of P. aeruginosa-induced biofilm, a 90.9% reduction in
CFU/biofilm, and (iii) in the case of S. aureus Mu50, a non-significant
reduction in single-species biofilms but a significant reduction (around
70%) in mixed-species biofilms. This enabled overcoming the difficulties
in treating chronic wound infections caused by multispecies biofilms
(Brackman et al., 2016). CD-mediated QSI has been observed to exploit
the high solubility of CD in water as a mechanism to effectively deliver
QS signal (AHL) analogs, which thus compete with AHL for receptor
sites. Here, inclusion into β-CDs inhibited P. aeruginosa virulence (host
evasion and biofilm development) in Caenorhabditis elegans (Ziegler
et al., 2021). These approaches will likely provide solutions that may be
superior to antibiofilm agents compared to conventional techniques,
such as antibiotics through wound care gauzes.
Extracts from Allium sativum L., Armoracia ruticana, Scutellaria bai­
calensis Georgi, and Phyllanthus amarus diterpene phytols have been
reported to inhibit biofilm formation by P. aeruginosa, Pectobacterium
carotovorum, and Vibrio campbellii (Kalia et al., 2019). The
indole-mediated inhibition of QS-regulated biofilm formation, EPS
production, and motility is owing to its effects on the three-channel QSS.
Microalgae are a source of auxins such as indole-3-acetic acid and
-acetamide. These biomolecules significantly reduce the pathogenicity
of V. campbellii. This leads to an enhancement in the survival rate of
Macrobrachium rosenbergii, a giant river prawn (Yang et al., 2017). Dioon
spinulosum extract against P. aeruginosa downregulated the expression of
the QS genes lasI, lasR, rhlI, rhlR, and ndvB, which resulted in the inhi­
bition of EPS production up to 51.4% and biofilm formation up to
77.1%. This process has applications in treating wounds and respiratory
and UTIs (Elekhnawy et al., 2022).
3.4. Anti-c-di-GMP-mediated biofilm formation
Targeting the c-di-GMP signaling system is a promising strategy for
developing antimicrobial drugs, primarily due to its effect on critical
bacterial lifestyle features without affecting their growth. Recent efforts
have focused on targeting bacterial pathogenicity and biofilm forma­
tion. Another feature that favors c-di-GMP as a drug target is its absence
in eukaryotes, reducing any potential threat to the infected host (Val­
entini and Filloux, 2019). Non-antimicrobial agents, including small
molecules, could inhibit the c-di-AMP-mediated transition of planktonic
to sessile lifestyles (Jenal and Dorman, 2009; Römling and Balsalobre,
2012). Recent works have focused on screening different compounds to
inhibit the functioning of c-di-GMP signaling. Thus, the inhibition of
biofilm formation and virulence factors were observed by (i) inhibiting
c-di-GMP synthesis through (a) DI-3 (N-4-anilinophenyl-benzamide) in
P. aeruginosa and Vibrio cholerae and (b) DI-8 (N-2-henylethyl-amino­
carbonothioyl-benzamide) in V. cholerae (Sambanthamoorthy et al.,
2012); (ii) reducing global levels of c-diGMP using (a) azathiopine in
E. coli and P. aeruginosa (Antoniani et al., 2013), (b) 6-mercaptopurine in
E. coli (Migliore et al., 2018), and (c) terrein in P. aeruginosa (Kim et al.,
2018); (iii) competing with c-di-GMP for its binding sites or its seques­
tration of (a) thiol-benzo-triazolo-quinazolinones in P. aeruginosa (Zhou
et al., 2013), (b) ebselen in P. aeruginosa (Lieberman et al., 2014), and
(c) proline-rich tetrapeptide Gup-Gup-Nap-Arg in P. aeruginoisa (Foletti
et al., 2018). Certain peptides structurally resemble effector proteins,
sequestering c-di-GMP, which inhibits biofilm formation by
V.C. Kalia et al.
P. aeruginosa (Hee et al., 2020). Diguanylate cyclases and degradative
phosphodiesterases also regulate the levels of c-di-GMP. Raffinose, a
galactotrisaccharide, inhibits biofilm matrix formation by enhancing the
activity of c-di-GMP-specific phosphodiesterase, thereby reducing the
concentration of c-di-GMP (Kim et al., 2016). Agents that reduce
c-di-GMP levels by activating esterases or inhibiting diguanylate cy­
clases can be envisaged as potential mechanisms to counter biofilm
formation. Diguanylate cyclase inhibitors include D-di-GMP and GTP. A
major limitation of this approach is the high variability between the two
enzymes (Cho et al., 2020). Anti-c-di-GMP peptides (YfiN interacting
peptides) have been identified in lytic bacteriophage PB1. These bind to
the diguanylate cyclase YfiN of P. aeruginosa and negatively impact the
functioning of c-di-GMP signaling, interfering with bacterial motility
and biofilm formation (De Smet et al., 2021).
3.5. Other approaches
3.5.1. Bacteriophages
BPs prevent biofilm formation by gram-negative and gram-positive
pathogenic bacteria. Screening of BP lysin CF-301 revealed that com­
plete eradication of S. aureus biofilms formed on the surfaces of catheters
and surgical meshes could be observed at ≤ 0.25 μg/mL within 1 h
(Schuch et al., 2017). Antibiotic-resistant Vibrio alginolyticus was
inhibited in the planktonic state by BP pVa-21 (Myoviridae). However,
up to 5 h is required to eliminate the bacteria present within the biofilm
(Kim et al., 2019). Biofilm formation by E. faecalis EF54 on natural tooth
roots was cleared by BP application (SHEF2). Furthermore, the survival
rate of zebrafish infected with E. faecalis strain OS16 was improved by
up to 84% (Al-Zubidi et al., 2019). Endolysin from BP vB_EfaS_PHB08
eradicated biofilms by reducing E. faecalis by 1 × 105 CFU. Thus, BPs
can treat pathogenic infections in wounds and the urinary tract (Yang
et al., 2020). The efficacy of BPs can be enhanced in combination with
antibiotics; biofilms caused by MSSA and P. aeruginosa in patients with
multi-organ failure on ventricular assistance are highly susceptible to
lytic BPs in conjunction with antibiotics. This has enabled successful
heart transplantations (Aslam et al., 2019). The lytic phage
ɸWL-3/fosfomycin combination was effective against
ciprofloxacin-resistant E. coli biofilms (Wang et al., 2020). The intra­
operative administration of BP cocktails along with antibiotics stopped
the recurrence of infections in patients with musculoskeletal infections
(Onsea et al., 2019). Recurrent urinary tract infections caused by the
XDR K. pneumoniae strain CX10301 were successfully treated with a
cocktail of lytic BPs and antibiotics (sulfamethoxazole-trimethoprim)
(Bao et al., 2020). Colonization of the gut by E. coli strain ST131-H30R
was successfully reduced by a cocktail of BPs and E. coli Nissle-1917,
which produces microcin C7 (Porter et al., 2022). A cocktail of lytic
BPs was used in phase I/II clinical trials to treat P. aeruginosa infections
in patients with burns. The trial was terminated prematurely because of
the poor efficacy of PP1131 (Jault et al., 2019).
3.5.2. Nanoparticles
A significant limitation of the use of antibiotics to treat pathogenic
bacterial biofilms is their burst release. NPs have proven effective in
achieving a sustained release of antibiotics (Al-Wrafy et al., 2022). The
primary advantage of NPs as carriers is targeted drug delivery. Coating
NPs with antibiotics enhances their stability and antimicrobial potency.
Enzymatic biofilm degradation can be achieved by targeting matrix
components of the matrixome (Karygianni et al., 2020a; b). Coating of
biopolymer NPs with amino-cellulose and hyaluronic acid inhibited
biofilm formation by up to 40% in E. coli and 94% in the case of S. aureus
(Ivanova et al., 2018) (Table 3). Multimetallic (ZnO:MgO) NPs are an
advancement for disrupting P. mirabilis cell membranes. These NPs
cause protein dysfunction and degrade biofilm DNA (Basavegowda and
Baek, 2021). Multi-functional ZnO-NPs at ½ × MIC of Zn-NPs produced
using phytocompounds (from Plumbago zeylanica) inhibited biofilm
formation by 60%, 70%, and 77.69%, and could also eradicate the
11
Ecotoxicology and Environmental Safety 264 (2023) 115389
preformed biofilms by 52.69%, 59.79%, and 65% in E. coli, S. aureus,
and P. aeruginosa, respectively (Husain et al., 2022). Selenium NPs as
intracellular drug delivery agents have shown superior QSI activity,
targeting the LasR receptor. Biofilm formation was inhibited > 90% at
4.5 μg/mL SeNPs@HP (Prateeksha et al., 2017). AgNPs coated with
thioether-bridged mesoporous organo-silica bind to ceftazidime, and
alginate lyase causes degradation of P. aeruginosa biofilms. In mouse
lungs the eradication of P. aeruginosa PAO1 was observed to be effective
with nanocomposites, via two routes (i) 68% and 85% inhibition and (ii)
47% and 72a% with Ag@MON-CE (NPs decorated with PEI (poly­
etherimide) loaded with ceftazidime) and Ag@MON-AE (NPs decorated
with alginate lyase and ceftazidime), respectively (Wan et al., 2020). Ag
and ZnO NPs altered biofilm formation and iron homeostasis in P. aer­
uginosa. AgNPs resulted in increased modifications in cell viability and
Reactive Oxygen Species production, leading to significantly high
disruption of the biofilm formation process. Cell viability decreased by
90% at 50 mg/L and 99.7% at 100 mg/L of ZnO NPs, whereas Ag NPs
did not show any significant changes. The impact on eDNA and carbo­
hydrate content of the biofilm matrix decreased by 32.82% and 59,92%
in the presence of 1 mg/L Ag NPs, respectively. These changes disturbed
the biofilm architecture (de Celis et al., 2022). Phloroglucinol encap­
sulated in chitosan NPs effectively inhibited biofilms formed by mono-
and dual-species of S. aureus, S. mutans, and K. pneumoniae. MIC values
of PG-CSNPs (10 mg/mL) against planktonic cells of S. aureus, S.
mutans, and K. pneumoniae were found to be 2048 µg/mL, whereas the
C. albicans was found to be higher than 2048 µg/mL. Sub-minimal
inhibitory concentrations (sub- MIC) of PG-CSNPs against preformed
mono/dual species biofilms was 256–2048 µg/mL Mature biofilm
formed by mono/dual species had an ½ BMIC80 AntiMicrobial
+ PGCSNPs of 1024 µg/mL (Khan et al., 2022). AgNPs synthesized using
an extract of Bothriochloa laguroides proved inhibitory to the
biofilm-forming abilities of S. aureus strains MRSA and MSSA (94%) and
Yersinia enterocolitica strains 8081 (91%) and ME110 (96%). An addi­
tional advantage is their potential to eradicate mature biofilms by 99%
for strain 8081, 94% for ME110 and 87% for MRSA and 82% for MSSA
(Toranzo et al., 2022). Niosome NP-encapsulated imipenem affects the
sensitivity and biofilms formed by S. epidermidis by reducing the
expression of icaD, FnbA, and EbpS (Piri-Gharaghie et al., 2022). The
essential oils at 200 mg/mL had strong antimicrobial activity against
P. mirabilis. Here, 92% deactivated survival rate with the Solanum nigrum
essential oils was recorded (Khaled et al., 2021). Inhibition of
A. baumannii biofilms was achieved with chitosan NPs loaded with
essential oil (150 µg/mL) (Govindan et al., 2022). Encapsulation of
alizarin liposomes in chitosan-gum arabic nanocarriers resulted in an
improved kinetic release mechanism. It efficiently inhibits 95% of the
biofilms formation by S. aureus (MRSA) MW2 and Candida albicans
DAY185 and ATCC10231 at CGL-Alz NCs (Alizarin: 50 μg/mL). The
effect was evident in mono- and dual species biofilms and cell aggre­
gation by C. albicans (Raj et al., 2022). The critical characteristics that
regulate the efficacy of NPs are their size, shape, surface charge, and
hydrophobicity. In contrast, the biofilm features that influence the ef­
ficiency of NPs are EPS matrix viscosity, compactness, liquid flow, and
other interactions (Robino and Scavone, 2020).
4. Stumbling blocks
Despite enormous research efforts to treat infectious diseases, there
are no assured treatment options. Antibiofilm agents that target their
formation and stability have been evaluated, and very few products have
been tested in clinical trials (Lu et al., 2022). There is a paucity of in vitro
studies on multi-species biofilms. Few studies have evaluated cytotox­
icity toward the host (Fleming and Rumbaugh, 2017). Another bacterial
characteristic that needs to be considered is their ability to switch back
to free-living planktonic entities owing to active self-dispersal. As
dispersed cells are more lethal than planktonic cells, it is important to
manage them post-biofilm disintegration. Thus, antibiofilm agents may
V.C. Kalia et al. Ecotoxicology and Environmental Safety 264 (2023) 115389

Table 3
Nanoparticles as therapeutic agents against biofilm-forming microbial pathogens.
Pathogen Inhibitor Support Mechanism Inhibitor concentration Applications Reference
(Source) (EC: Effective; AC: Applied)

Staphylococcus Amino-cellulose Nanoparticles Interacts with EC: 4 × 1010 NPs/mL Inhibit biofilm Ivanova et al.
aureus, and hyaluronic (NPs) phospholipids of the AC: 1 × 1010, 2 × 1010, and formation in E. coli (2018)
Escherichia coli acid (Biocompatible bacterial cell membrane 4 × 1010 NPs/mL (40%) and S. aureus
polymer) (94%). It did not
influence the human cell
viability
S. aureus, E. coli, Plumbago Zinc oxide (ZnO)- Effects EC: ½ × MIC of Zn-NPs Inhibit biofilm Husain et al.
Pseudomonas zeylanica NPs Exopolysaccharide AC:1⁄₁₆, 1/8 and ¼ × MIC of ZnO- formation (up to 77%) (2022)
aeruginosa (extract) production NPs, and eliminate
MIC: E. coli, and S. aureus 200 µg/ preformed biofilms (up
mL, to 65%
P. aeruginosa 400 µg/mL
S. aureus, S. mutans, Phloroglucinol Chitosan NPs Interact with the EC: PG-CSNPs at 1024 μg/mL for Promotes antibacterial Khan et al.
Klebsiella negatively charged sugar dual sp. biofilm (BF); 256 μg/mL activity 86–92% BF (2022)
pneumoniae, polymers and eDNA of against C. albicans; 512 μg/mL inhibition. Prevent the
Candida albicans the ‘matrixome’ against S. mutans and S. aureus; spread of the epidemic
1024 μg/mL against Klebsiella and treat mixed-species
pneumoniae biofilms
AC: Preformed BF: PG-CSNPs
(10 mg/mL)
Sub-MIC 256–2048 µg/mL
Mature BF: ½ BMIC80
Antimicrobial + PGCSNPs
1024 µg/mL
S. aureus, Yersinia Bothriochloa Gold NPs (AgNPs) Inhibited planktonic and Biofilm inhibition: Potential for developing Toranzo et al.
enterocolitica laguroides sessile growth EC: 125 pmol/L (8 × MIC) for a new class of (2022)
(extract) Y. enterocolitica strains; 250 pmol/ antibacterial agents
L for S. aureus strains
Mature biofilm eradication:
EC: 500 pmol/L (30 × MIC) for
Y. enterocolitica strains; 250 pmol/
L for S. aureus strains
AC: 16 pmol/L for Y. enterica and
64–125 pmol/L for S. aureus
S. aureus, C. Alizarin Gum arabic Negatively charged gum Biofilm inhibition (Mono/Dual): Inhibit biofilm Raj et al. (2022)
albicans chitosan Arabic with improved EC: CGL-Alz NCs (Alizarin: 50 μg/ formation of
kinetic release mL). multispecies pathogens
AC: MIC: C. albicans 400 μg/mL,
and S. aureus 600 μg/mL; CGL-Alz
NCs (Alizarin: 0–50 μg/mL).
P. aeruginosa Polyphenols from Selenium NPs Affect QS-mediated Biofilm formation Anti-virulence activities Prateeksha
honey pathogenicity EC: 4.5 μg/mL, SeNPs@HP, (in-vitro and in-vivo) et al. (2017)
AC: Chromobacterium violaceum
CV12472 − 10 μg/mL; P.
aeruginosa PAO1–15 μg/mL
P. aeruginosa Thioether- Silver NPs (AgNPs) Binding to ceftazidime Biofilm formation and Inhibition of biofilm Wan et al.
bridged and alginate lyase Degradation: formation and rapid (2020)
mesoporous EC: ceftazidime: 0.656 mg/mL degradation of biofilms
organo-silica Ag@MON-CE/AE - 1.72 mg/mL in mouse lungs.
AC: ceftazidime: 0.27–0.656 mg/ Reduced lung injuries
mL and no deaths or serious
Ag@MON-CE/AE - 0.07, 0.14, side effects
0.21, 0.43, 0.86, and 1.72 mg/mL
P. aeruginosa. NPs Ag- and ZnO-NPs Effect growth, biofilm EC: Cell viability at ZnO 100 mg/ Higher disruption in de Celis et al.
formation, ROS L; biofilm formation (2022)
production, iron eDNA and carbohydrates by 1 mg/
homeostasis, eDNA L Ag NPs.
AC: Ag (1 and 5 mg/L) and ZnO
(50 and 100 mg/L) NPs
Proteus mirabilis NPs ZnO:MgO-NPs Disrupt cell membranes; EC: No specific information Biofilm degradation Basavegowda
protein dysfunction and AC: No specific information and Baek
degrade biofilm DNA (2021)
Acinetobacter Plant essential Chitosan NPs Effective removal of EC: 150 µg/mL Effective against Govindan et al.
baumannii oils biofilm formation AC: 150 µg/mL nosocomial and urinary (2022)
tract infections
P. mirabilis Essential oils None Effect survival and EC: 200 µg/mL Active against urinary Khaled et al.
(Solanum nigrum) virulence factors AC: 25–200 µg/mL tract infectious bacteria (2021)

PG-CSNPs: phloroglucinol-chitosan nanoparticles; sub-MIC: Sub-minimal inhibitory concentrations.

Ag@MON-AE: NPs decorated with alginate lyase and ceftazidime, while Ag@MON-CE: Ag@MON-PEI (polyetherimide) loaded with ceftazidime

12
V.C. Kalia et al.
be supplemented with other bioactive molecules such as nitrous oxide
(from sodium nitroprusside) (Uppuluri and Lopez-Ribot, 2016). The use
of QSIs as antipathogens interferes with biofilm formation and patho­
genicity without killing the pathogens. Therefore, free-living cells can be
exposed to QSIs along with low doses of antibiotics, reducing the risk of
the emergence of drug-resistant microbes (Kalia et al., 2019). The
integration of biological and in silico techniques may facilitate the future
design of antibiofilm agents (An et al., 2021).
5. Conclusions
Targeting the biofilm formation process is more desirable than tar­
geting fully formed biofilms for the treatment of infectious diseases. This
scenario is more complex than is envisaged. Coating medical equipment
with biomolecules, including QSIs, that restrict bacterial adhesion,
biofilm formation, and disruption of mature biofilms has been proposed.
Antibiofilm-forming agents have gained importance because of their
wide range of applications in aquaculture, water purification, waste­
water treatment, shipping, agriculture, and the maintenance of
archaeological monuments. Despite the promising discovery of anti­
biofilm agents, tremendous efforts are required to translate their po­
tential for the treatment of infectious diseases.
CRediT authorship contribution statement
Vipin Chandra Kalia: Conceptualization, Writing – original draft
preparation, Visualization, Writing – review & editing. Sanjay Kumar
Singh Patel: Writing – original draft preparation. Jung-Kul Lee: Writing –
review & editing, Resources, Funding acquisition, Supervision, Project
administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
I have mentioned all the references.
Acknowledgements
This research was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF), funded by
the Ministry of Science, ICT & Future Planning (NRF-2022M3A9I30823
66, NRF-2021R1A2B5B03002728, and 2022M3A9I5015091). The
sponsor(s) had no role in the study design; in the collection, analysis,
and interpretation of data; in the writing of the report; and in the de­
cision to submit the article for publication.
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