International Journal of Medical Microbiology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Medical Microbiology

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

Biofilms: Survival and defense strategy for pathogens

⁎
Ashutosh Kumara,1, Anwar Alama,1, Mamta Ranib, Nasreen Z. Ehteshamc, Seyed E. Hasnaina,d,e,
a
Molecular Infection and Functional Biology Lab, Kusuma School of Biological Sciences, Indian Institute of Technology-Delhi, New Delhi, India
b
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
c
National Institute of Pathology, Safdarjang Hospital Campus, New Delhi, India
d
JH-Institute of Molecular Medicine, Jamia Hamdard, New Delhi, India
e
Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India

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

Keywords: Studies on biofilm related infections are gaining prominence owing to their involvement in majority of clinical
Biofilms infections. Biofilm, considered as a generic mechanism for survival used by pathogenic as well as non-pathogenic
Drug tolerance microorganisms, involves surface attachment and growth of heterogeneous cells encapsulated within a matrix.
Microbiota The matrix provides ecological niche where partnership of cells endows a community like behaviour that not
Mycobacteria
only enables the cohort to survive local microenvironment stress but also channelizes them to evolve, dis-
Biofilm regulators
Biofilm disrupters
seminate and cause resurgence of infections. In this mini-review we highlight the mechanisms used by microbes
to develop and sustain biofilms, including the influence of the microbiota. Several strategies to target biofilms
have been validated on certain groups of microorganisms and these basically target different stages in the life
cycle of biofilm, however comprehensive methods to target microbial biofilms are relatively unknown. In the
backdrop of recent reports suggesting that biofilms can harbour multiple species of organisms, we need to relook
and devise newer strategies against biofilms. Effective anti-biofilm strategies cannot be confined to a single
methodology that can disrupt one pathway but should simultaneously target the various routes adopted by the
microorganisms for survival within their ecosystem. An overview of the currently available drugs, their mode of
action, genomic targets and translational therapies against biofilm related infection are discussed.

1. Introduction
Majority of the microorganisms develop different types of survival
mechanisms such as growth regulation, heterogeneity in population,
proteolytic systems etc. to adapt to stress conditions. Pathogenic mi-
croorganisms acquire the ability to sustain various host immunological
responses. Biofilms are heterogeneous congregation of surface asso-
ciated microorganisms encapsulated within a self-produced polymer
matrix consisting of polysaccharide, protein and DNA. Several factors
such as bacterial defense mechanism, suitable area for colonization, the
community cooperation related benefits and extraordinary mode of
growth in their habitat accelerate biofilm formation (Jefferson, 2004).
The matrix acts as a physical barrier against drugs and provides a
protective ecological niche for the survival of microorganisms. A single
species of bacteria or consortia of multispecies microbes can exist
within a biofilm. Biofilm associated diseases caused by several species
of microorganisms such as Mycoplasma pneumoniae, Candida albicans (C.
albicans), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus epi-
dermidis, Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium
abscessus (M. abscessus) etc., pose serious health concerns due to re-
calcitrant nature of microbes towards antimicrobial drugs and host
immune responses.
Biofilms are a major health concern contributing to nearly 80% of
the recalcitrant hospital infections (Davies, 2003). Bacteria such as
Staphylococcus aureus (S. aureus), P. aeruginosa, Acinetobacter baumannii
and other clinically important microorganisms that thrive on medical
devices form biofilms which confer them with up to 1000 times more
resistance and tolerance to antibiocides as compared to their planktonic
forms. Nearly 80% of the pathogenic bacteria are associated with
medical device related infections such as ortho-dental prosthetics,
contact lenses, cardiovascular valves, urinary catheters, pacemakers
and breast implants (Scott, 2009). Bacteria can also modulate the pH of
the environment in order to facilitate the formation of biofilm. Klebsiella
and Pseudomonas increase the alkalinity of urine in order to form bio-
films on urinary catheters. These biofilms cause secondary complica-
tions when they detach from the catheter causing tissue injury in ur-
inary bladder (Neethirajan et al., 2014).
Biofilm formation by M. tuberculosis for its survival within the host

⁎
Corresponding author at: Molecular Infection and Functional Biology Lab, Kusuma School of Biological Sciences, Indian Institute of Technology-Delhi, New Delhi, India.
E-mail addresses: seyedhasnain@gmail.com, seh@bioschool.iitd.ac.in (S.E. Hasnain).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ijmm.2017.09.016
Received 14 January 2017; Received in revised form 11 September 2017; Accepted 19 September 2017
1438-4221/ © 2017 Elsevier GmbH. All rights reserved.

Please cite this article as: Kumar, A., International Journal of Medical Microbiology (2017), http://dx.doi.org/10.1016/j.ijmm.2017.09.016
A. Kumar et al. International Journal of Medical Microbiology xxx (xxxx) xxx–xxx

may be one of the main cause behind long term drug treatment of tu-
berculosis patients (Hopewell et al., 2006, 2014). The involvement of
non-tuberculous mycobacteria (NTM) such as M. abscessus in biofilm
formation in cystic fibrosis (CF) patients has been reported (Qvist et al.,
2013). CF is caused by mutation in the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) gene which is present on the long arm
of chromosome 7. The mutated/abnormal CFTR protein (a cAMP-acti-
vated ion channel) leads to decreased chloride secretion and increased
sodium absorption across the epithelial surface. This results in thick-
ened mucus secretions in lung airways where bacteria are trapped and
colonize. M. abscessus exists in two phenotypic variants; the smooth
form expresses glycopeptidolipids whereas the rough form expresses
minimal amounts of glycopeptidolipids (Howard et al., 2006). Different
studies have confirmed that smooth variant persists inside biofilm
whereas rough variant can cause persistent and acute infection in the
host (Catherinot et al., 2009; Bernut et al., 2014). M. abscessus can
convert itself bidirectionally into smooth and rough forms which have
been shown in vitro by routine plating (Byrd and Lyons, 1999; Howard
et al., 2006). This ability of M. abscessus perhaps allows it to colonize as
a smooth-morphotype in the lung airways of CF patient and later cause
acute respiratory failure upon emergence of an isogenic rough-mor-
photype (Catherinot et al., 2009).
Besides these, the microbiota flourishes along the epithelial walls or
exposed tissue surface and locally form biofilms inside the host body
within wounds, urogenital system, respiratory tract or as dental pla-
ques. Such biofilms, besides causing local infections also predispose
individuals to secondary long term manifestations. For example,
Porphyromonas gingivalis causes periodontal biofilms within dental
plaques that also play a major role in the progression of cardiovascular
diseases (Leishman et al., 2010). Several clinically relevant biofilm
forming bacteria are listed in Table 1.
2. Biofilm stages
Bacterial biofilms can form on abiotic surfaces as well as on biotic
surfaces and proceed through distinct stages which can be broadly ca-
tegorized into reversible adhesion stage and irreversible cohesion stage.
In vitro models of biofilm formation does not necessarily represent the
outcome in in vivo models due to the difference in host environment
where the challenges are different in terms of nutrient availability,
immune stress etc. Microbial cells within the biofilm evolve multiple
mechanisms to proceed through the four stages of biofilm development,
namely, (i) attachment to surface, (ii) sessile growth, (iii) colonization,
and (iv) dispersal (Fig. 1). Unique set of genes are expressed in each
stage of biofilm formation that enable production of extracellular ma-
trix consisting of polysaccharides, glycopeptidolipids, proteins and fatty
acids. During the initial stages of reversible adhesion, bacterium can
aggregate loosely but can dissociate and revert back to planktonic
forms. The initial stages of abiotic biofilm formation are driven by
electrostatic interactions between the substratum and the bacteria,
surface topography and are facilitated by the presence of adhesins on
bacterial pili. The probability of making initial interaction within the
substratum is influenced by hydrodynamic forces arising from the
viscosity of medium and presence or absence of chemotactic molecules.
Antigen 43 of uropathogenic Escherichia coli (E. coli) mediates attach-
ment to the abiotic surface and also aids in intercommunication be-
tween the bacteria of same species (Ulett et al., 2007). Mutant strains of
P. aeruginosa lacking CheR1 methyltransferase fail to express chemo-
tactic amino acids that are required for surface sampling, attachment
and maturation of biofilm (Schmidt et al., 2011). Flagellate bacteria can
encounter hydrodynamic stress and hence have the advantage to form
biofilms. In the case of biotic surfaces, attachment of bacteria to the
host surface is largely through the interaction of bacterial proteins with
the extracellular proteins or carbohydrate moieties on the surface of the
cells/tissues and depends on the hydrophobicity of interacting cell
membranes. In P. aeruginosa, surface pili type IV aids in adherence and
movement through the viscous cell surface (Klausen et al., 2003). Pili
type I adhesins, e.g. FimH, bind to mannosylated moieties on host cells
and have been implicated in pathogenesis caused by uropathogenic E.
coli (Wright et al., 2007). Several other types of adhesion molecules,
such as Sag, have since been reported in Enterococcus spp. that bind to
collagen of eukaryotic cells and provide focal points for aggregation
and adherence to eukaryotic cells, leading to biofilm formation
(Mohamed et al., 2006). S. epidermidis and S. aureus express microbial
surface components recognizing adhesive matrix molecules that attach
non-covalently to fibronectin, fibrinogen and human matrix proteins
(Fey and Olson, 2010). Upon contact with the surface, auto-inducer
signals initiate the switchover of gene expression that leads to upre-
gulation of factors required for sessile colony formation. Master reg-
ulator switches, such as PrfA and SinR, have provided insights into
mechanisms regulating genetic reprogramming in Listeria mono-
cytogenes and Bacillus subtilis biofilms, respectively (Luo et al., 2013a,b;
Newman et al., 2013). The role of small non-coding RNAs (sRNAs) in
regulating the switch between planktonic to biofilm mode and vice
versa is evident from the observation that sequestration of RNA reg-
ulatory proteins, such as RsmA with sRNAs, switches development of
planktonic form into biofilm stages in P. aeruginosa (Ventre et al.,
2006).
Attachment of Mycobacterium smegmatis to the substratum induces
synthesis of alginate, glycopeptidolipid and small chain mycolic acids
aided by the activity of chaperonic proteins such as GroEL-1 (Davies
et al., 1993; Recht and Kolter, 2001). Mycolic acids, being hydrophobic,
allow non-covalent binding with host cell proteins such as fibronectin,
fibrinogen, collagen IV etc. Alteration in mycolic acid synthesis through
disruption of polyketide synthatase affects both formation of biofilm
and virulence (Ojha et al., 2008; Pang et al., 2012). The attractive or
repulsive phenomenon between bacteria in the niche depends on the
availability of nutrients, pH, presence of ions and temperature. Once
the bacterium attaches to the surface they interact with other bacteria
of the same species or other species which initiates the process of

Table 1
List of clinically relevant biofilm forming bacteria.

Location Organism Reference

Lungs of patients with Cystic Fibrosis Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenza, Burkholderia cepacia complex Lopes et al. (2015)
Pacemakers Staphylococci, Streptococci Greenspon et al. (2008)
Prosthetic heart valves Staphylococcus epidermidis, Staphylococcus aureus, Streptococci, Gram-negative bacilli, Diphtheroids, Enterococci Donlan (2001)
and Candida spp.
Wounds (equine) Staphylococci, Bacillus, Enterococci, Pseudomonas, Clostridium etc. Westgate et al. (2011)
Wounds (dog) Staphylococcus intermedius, Staphylococcus epidermidis, Streptococcus canis Swanson et al. (2014)
Contact Lenses Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, El-Ganiny et al. (2017)
Klebsiella spp.
Orthopaedic implants Staphylococci, Pseudomonas genus, Enterococci, Streptococci Arciola et al. (2015)
Breast implants Staphylococcus epidermidis, Pseudomonas aeruginosa Seng et al. (2015)
Dental Streptococcus mutans, Streptococcus sanguinis, Porphyromonas gingivalis, Fusobacterium nucleatum, Lactobacillus Huang et al. (2011)
casei, Actinomyces naeslundii

2
A. Kumar et al.
cohesion or attachment among microbial cells to form microcolonies.
Consequent to these events, bacteria are irreversibly attached to the
substratum and can overcome hydrodynamic stresses by virtue of their
colonization and maturation. Motility of bacteria is reduced and the
cells secrete exopolysaccharides that aid in entrapping nutrients.
Transition of single cell into group of cells necessitates the need to
develop intercommunication so as to coordinate growth and maximize
efficiency. Bacteria coordinate growth through quorum sensing system
using several signalling peptides and molecules such as competence
stimulating peptide, 3,5-cyclic diguanylic acid (c-di-GMP), farnesol,
rhamnolipids, phenol-soluble modulins etc. Quorum sensing helps to
maintain optimum cell density as high as 107cells/cm2, required for
virulence of Vibrio chlorea (Deep et al., 2011). Quorum sensing in gram
positive and negative bacteria is predominantly mediated through small
peptides and acyl-homoserine lactones, respectively (Taga and Bassler,
2003). In C. albicans, high levels of farnesol inhibit mycelium conver-
sion and facilitate active division by budding (Hornby et al., 2001). In
P. aeruginosa, rhamnolipids are required for intercellular interactions
and maintenance of open channels between cellular aggregates that aid
in distribution of nutrients (Davey et al., 2003).
Studies in uropathogenic E. coli show that cellulose and curli amy-
loid fibers are essential for pellicle formation (Cegelski et al., 2009). In
patients suffering from CF, P. aeruginosa upregulates proteins such as
Pel that play crucial role in attachment to the epithelial surface. During
the colony formation stage new set of proteins such as Psl and CdrA
adhesins are upregulated in response to high levels of signalling mo-
lecules, c-di-GMP, which reinforce matrix components and stabilize
pellicle formation (Ma et al., 2006; Borlee et al., 2010). Several proteins
associated with motility and fimbriae formation, lipopolysaccharide
synthesis and cell wall surface are involved in biofilm formation in
bacteria (Niba et al., 2007; Cucarella et al., 2001; Geoghegan et al.,
2010).
Colonization of bacteria induces secretion of exopolysaccharides,
proteins, extra-cellular DNA (eDNA) etc. that not only aid in structural
stability but also enhance substrate exchange and distribution of nu-
trients. The components of the matrix vary according to the bacterial
species involved in biofilm formation. The genes on plasmids encoding
biofilm proteins are horizontally transferred and aid in transmission of
3
International Journal of Medical Microbiology xxx (xxxx) xxx–xxx
Fig. 1. Combination of antibiotic and biofilm dis-
ruptive agents modulate biofilm developmental
stages. Microorganisms undergo four definitive
stages for completion of biofilm cycle involving ad-
herence, sessile growth, colonization and dispersal.
Biofilm matrix impedes entry and interferes with the
interaction of antibiotic with the cell. Strategies di-
rected against biofilm formation involve use of an-
tibiotics in combination with biofilm disrupters,
specific for developmental stages (i–iv). Attachment
of planktonic form to the surface (i) can be in-
capacitated by using silver coatings. Microbial com-
munities coordinate using quorum sensing (QS) and
grow as sessile structure (ii) encapsulated within a
matrix. Use of inhibitors (lactoferrin, DNase,
Clarithromycin) against quorum sensing molecules
and matrix components disrupts sessile growth.
Colonization of persister cells require transcription of
genes – PKA and PNAG, (iii) which may be silenced
using siRNA. Nutritional limitations induce stress
like conditions and favour dispersal of persister cells
(iv). Use of biofilm nano-disrupters (Fe3O4, D-amino
acids) and stress inducers (NOS) can facilitate dis-
ruption of matrix allowing antibiotics to enter the
biofilm shield and act on cells.
virulence factors. Nutrient availability is an important determinant in
shape and size of biofilm. A gradient in nutrient and oxygen from the
outer to the inner core causes variability in metabolic activity of cells
and biodiversification. The outer layer of cells grows faster as compared
to the inner core. In such an environment, starved cells achieve static
growth and hence their numbers get depleted. Such dormant cells
achieve antibiotic tolerance and are difficult to detect in chronic biofilm
infections. Sub-inhibitory concentrations of Imipenem or Tobramycin
may induce alginate production in P. aeruginosa (Bagge et al., 2004).
The hallmark of biofilm formation is to achieve greater co-ordination.
Cells in the outer layer of C. albicans express genes associated with
synthesis of proteins required for glycolysis, cholesterol and cell wall
whereas cells in the inner core express genes associated with enzymes
required for sulphur metabolism or degradation of the cell wall. This is
indicative of the fact that biofilm is an active congregation of cells that
have attained the sufficiency to modulate their metabolic engine as
required in response to the microenvironment challenge.
The last phase of biofilm culminates into detachment of cells that
causes dissemination of single or clustered cells. The dispersal of the
bacterium from the biofilm is not a passive process, rather it is a highly
orchestered event that leads to release of specific cells at the final stages
of biofilm life cycle. Cells along the inner core of the biofilms lyse prior
to their dispersal. These lysed cells provide nutrients to the bacterium
that would undergo dispersal at a later stage. The cells undergoing
dispersal shut down the genes encoding exopolysaccharides or fimbriae
and simultaneously upregulate genes encoding chemotactic proteins or
flagella required for planktonic lifestyle (Rollet et al., 2009).
3. Biofilm components
The composition of the matrix has been studied in bacteria such as
P. aeruginosa, Bacillus spp., Staphylococcus spp. and Streptococcus spp.
(Lopez et al., 2010). The constituents of extracellular matrix depend on
the environment and the bacteria present within the biofilm (Fig. 2).
During the process of transition from a free living planktonic stage into
a congregation of cells, bacteria require greater coordination to attain
maximal efficiency within the limited space and nutrition. Although
quorum sensing molecules are secreted by free living bacteria as well,
A. Kumar et al.
phylococcus aureus consists of polymer of N-acetyl glucosamine, adhesive proteins and extracellular DNA.
these molecules hold greater significance in biofilm biology as they are
considered viable targets for drugs and could play a crucial role in
tackling biofilms. In P. aeruginosa, rhanmnolipids not only act as
quorum sensor but also block chemotactic activity of polymorpho-
nuclear (PMN) cells and induce necrosis. This is evident from the fact
that newly established biofilms are more susceptible to PMN attack as
compared to mature biofilms (O’Loughlin et al., 2013). The main con-
stituents of biofilm produced by P. aeruginosa are polysaccharides such
as Psl, Pel and alginate, that increase extracellular polymeric matrix
hydration and decrease flexibility of biofilm (Orgad et al., 2011). Many
proteins identified in biofilms are associated with outer membrane of
vesicle and are either secretory or cytoplasmic in nature (Toyofuku
et al., 2012).
The polymeric components of extracellular matrix entrap alginate,
extracellular amyloids, iron, pili etc. which contribute to adherence,
hydration, rigidity and matrix formation. Consequently, the extra-
cellular matrix provides physical barrier and subdue direct effects of
antibiotics on bacterial cells (Orgad et al., 2011; Alsteens et al., 2012;
Ramsugit et al., 2013). The recalcitrant nature of biofilm is a cumula-
tive effect of inertness of polymeric extracellular components and
adaptability of bacteria against antibiotics. Physical barrier of matrix
leads to a decreasing gradient of drugs along the outer surface towards
inner core of biofilm (Stewart, 2003). Bacteria depend on host iron to
upregulate polysaccharide intercellular adhesin (PIA) molecules re-
quired for fibrin mediated biofilm formation on implanted biomaterials
(Lin et al., 2012). Lactoferrin, an iron binding protein, reduces iron
supply necessary for the survival of bacteria. Bacteria subvert the effect
of lactoferrin by secretion of proteases against lactoferrin (Schmidtchen
et al., 2002).
4. Biofilm regulators
The master regulatory proteins trigger genes that control expression
of factors required for biofilm formation. Several master regulator
genes such as transcription activator PrfA in Listeria monocytogenes and
flagella regulator SinR in Bacillus subtilis have been identified that play
crucial role in biofilm (Luo et al., 2013a,b; Newman et al., 2013). These
changes in gene expression pattern weaken the argument that biofilm
formation is part of developmental stage utilized by bacteria to com-
plete their life cycle. Rather it is now apparent that biofilm formation is
a process by virtue of which microorganisms alter their phenotype to
adapt to environmental stresses or immune responses.
Transcriptomic analysis of genes expressed during various stages of
biofilm formation fails to provide a consensus pathway due to the im-
mense physiological heterogeneity and diversity of species harboring
the biofilm. The consistent factor in the regulation of biofilms is c-di-
GMP that helps in aggregation of cells and a decrease in its level causes
dispersal of cells from biofilms. The process of dispersal of bacteria from
the biofilms is also regulated. In Pseudomonas putida, diminishing levels
of c-di-GMP triggers cysteine proteinase activity that degrades
4
International Journal of Medical Microbiology xxx (xxxx) xxx–xxx
Fig. 2. Extracellular matrix of bacterial biofilms:
Extracellular matrix mainly consists of extracellular
polysaccharides, proteins and extracellular DNA that
are essential for the formation and maintenance of
biofilms. The nature and composition of biofilms
vary between different species and also within
strains of same species and with the environment
(Lopez et al., 2010). The composition of extracellular
matrix of three bacterial species Pseudomonas aeru-
ginosa, Bacillus subtilis and Staphylococcus aureus are
well studied. Extracellular matrix of Pseudomonas
aeruginosa consists of exopolysaccharides, surface
proteins, lectin binding proteins and extracellular
DNA. Extracellular matrix of Bacillus subtilis consists
of exopolysaccharide, Poly-gamma-Glutamate and
extracellular proteins. Extracellular matrix of Sta-
membrane protein LapG, causing dispersal of cells. P. aeuroginisa uti-
lizes rhamnolipids to cause dispersal of Klebsiella pneumonia, S. aureus
and C. albicans (Davies and Marques, 2009).
5. Biofilm in drug tolerance and persister formation
Within biofilm, cells exhibit morphological, replicative and phy-
siological heterogeneity aided by differential gene expression due to the
gradient in diffusible gases, toxic components or nutritional pressure
(Stewart, 2003). Depleted nutrient and oxygen within biofilm promote
asynchronous growth of non-replicative cohorts which exhibit fluc-
tuations in gene expression and may lead to drug tolerance. Phenotypic
diversity of bacteria within biofilm not only enhances greater co-
ordination but also empower them to reprogramme genes required for
efflux of toxins, DNA repair, ion sequestration, lipid biosynthesis, host
immune modulation etc., which in turn provides persistence and se-
lective propagation of resilient cells enduring stress (Anderson et al.,
2003; Lewis, 2005; Fux et al., 2005; Lebeaux et al., 2014).
Within biofilms, fraction of bacteria evolve as persister cells that are
genetically similar but are physiologically different compared to parent
cells. Persisters are metabolically inert, replicate slowly, modulate
toxin-antitoxin system, upregulate DNA repair and anti-oxidative ma-
chinery, have enhanced phosphate metabolism, and exhibit unrespon-
siveness towards minimal inhibitory concentrations of antibiotics
(Lewis, 2010). Drug treatment normally kills planktonic cells and the
majority of biofilm cells. However, drug tolerant persisters repopulate
the biofilm, disseminate into planktonic forms and start a new cycle of
biofilm development (Lewis, 2010; Zhang, 2014; Keren et al., 2011)
that increases the duration of treatment of diseases caused by biofilm
forming pathogenic microorganisms (Fig. 3). Low oxygen condition
induces biofilm whereas normoxia decreases biofilm formation (Totani
et al., 2017). Enhanced bacterial respiration reduces the persisters in
bacterial population (Vilchèze et al., 2017).
Toxin-antitoxin systems that may control bacterial growth and
metabolism induce the persister phenotype, pointing to the role of
several other growth regulatory proteins of M. tuberculosis in drug tol-
erance (Wang and Wood, 2011; Kumar et al., 2017). Mycobacterial
proteins that inhibit translation by interacting with ribosome (Trauner
et al., 2012; Kumar et al., 2012; Li et al., 2015) possibly induce persister
phenotype and drug tolerance because it has been reported that protein
synthesis inhibition by tetracycline induces persisters cells and aids in
survival (Kwan et al., 2013). The M. tuberculosis PpiB found in the se-
cretory proteome and membrane fraction (González-Zamorano
et al.,2009; Xiong et al., 2005; Souza et al., 2011) is also involved in
biofilm formation. PpiB can be a viable target for developing new
strategies for intervention against drug resistant TB (Pandey et al.,
2016; Hasnain et al., 2017).
A. Kumar et al.
Fig. 3. Mechanism of drug tolerance through biofilm: Fractions of bacteria in biofilms are
present as persisters that enhance drug tolerance. Drug treatment normally kills plank-
tonic cells and the majority of biofilm cells and when drug concentration drops, persister
bacteria re-populate the biofilm due to their drug tolerance nature. Hypoxia enhances
pellicle formation whereas normoxia decreases pellicle formation (Totani et al., 2017).
Enhanced bacterial respiration due to presence of cysteine or other small thiols causes
decrease in persisters/drug tolerance in bacterial population (Vilchèze et al., 2017). The
reduction in drug tolerance of bacterial infection shortened disease treatment duration.
Verified and putative roles are shown with solid and dotted lines, respectively.
6. Evasion of immune responses
Adherent bacteria cannot be opsonised easily and block signalling in
polymorphonuclear leukocytes (Gunther et al., 2009; Chorell et al.,
2012; Piatek et al., 2013). In patients with CF, P. aeruginosa produces
alginate and rhamnolipids to evade macrophages and induction of ne-
crosis in polymorphonuclear leukocytes, respectively (Leid et al., 2005;
Van Gennip et al., 2009). S. aureus escapes phagocytosis in blood by
producing coagulase that activates prothrombin and polymerizes
monomeric fibrinogen into insoluble fibrin, a constituent of biofilm
matrix (Cheng et al., 2010). Upon contact with macrophages, S. aureus
biofilms release lytic toxins that skew differentiation of activated
macrophages into M2 lineage. The defection towards M2 lineage re-
duces inducible nitric oxide synthase (iNOS) and increases arginase 1
(enzyme involved in collagen synthesis), consequently leading to fi-
brosis and evasion of recognition by Toll-like receptors (Hanke and
Kielian, 2012).
Cells within biofilms monitor the microenvironment within and
outside the physical boundaries to evade host immune responses or
utilize the host machinery to propagate. Biofilms of M. tuberculosis
develop within host tissues to survive the prolonged drug challenge in
TB patients. NTMs, such as M. abscessus and Mycobacterium avium, cause
secondary manifestations in CF patients (Hopewell et al., 2006, 2014;
Qvist et al., 2013). Within the macrophage, M. tuberculosis requires
eukaryotic serine/threonine protein kinase G (PKG) to evade fusion
with the lysosome. PKG associates with mycobacterial nudix hydro-
xylase (RenU) and accelerates hydrolysis of NADH or FAD which
maintains an optimum redox potential in macrophage. Disruption of
PKG-RenU pathway impairs mycobacterial sensitivity to oxidative
stress, impairs biofilm growth and reduces survival of M. tuberculosis
within macrophages (Wolff et al., 2015).
Staphylococcus epidermidis biofilm interferes with the activation of
5
International Journal of Medical Microbiology xxx (xxxx) xxx–xxx
complement proteins C3b or deposition of IgG on the surface of the
bacterium and provides resistance to phagocytosis by neutrophils. This
has a positive implication because inhibition of biofilm formation could
render the pathogen susceptible to complement mediated lysis (Kristian
et al., 2008). eDNA, generated by autolysis of bacteria, helps in colo-
nization of sessile form, suppresses host innate immune response, in-
creases tolerance to antibiotics and aids in virulence (Nguyen et al.,
2010; Qin et al., 2007; Madsen et al., 2012; Thurlow et al., 2011; Rose
et al., 2015). A combination of DNase with antibiotics such as AM-228,
Moxifloxacin or Clarithromycin, have been shown to be more effective
in mycobacterial killing as compared to antibiotic alone, suggesting
that eDNA can be a suitable target against biofilm formation (Aung
et al., 2016).
Polymicrobial interactions can be either synergistic or antagonistic.
C albicans can coaggregate symbiotically with S. aureus such that the
Candida hyphae penetrate the epithelial barrier and provide route for
entry of S. aureus. The symbiotic association increases virulence and
metabolic cooperation (Peters et al., 2010).
7. Anti biofilm approaches
It has been noted that antibiotic tolerant bacteria residing within
biofilms become susceptible to antibiotics upon dispersal from biofilm
suggesting that resilience towards antibiotics is due to phenotypic
adaptability and not essentially due to genetic adaptability (Fig. 1)
(Anwar et al., 1989). Factors such as mechanical stress, enzymatic di-
gestion, pH, oxygen availability, temperature and limiting nutrition
trigger dispersal of cells from the biofilm. Use of silver coating on
medical devices prevents surface attachment of bacteria. Clari-
thromycin blocks biofilm matrix formation in P. aeruginosa (Yasuda
et al., 1993). Ciprofloxacin reduces the overall thickness of the biofilm
and exposes the immature biofilm to phagocytosis by polymorpho-
nuclear neutrophils. Streptokinase dissolves the matrix polymer of
biofilm in S. aureus (Nemoto et al., 2000). Acyl-homoserine lactone
interferes with cellular signalling mechanisms used for quorum sensing
and affects normal biofilm formation (Parsek and Greenberg, 2000).
Epigallocatechin gallate and polyphenol in green tea remodels amyloid
fibrils in P. aeruginosa biofilm and increases the antibiotic potency
(Stenvang et al., 2016). As a result, immune cells can easily phagocy-
tose bacterial cells or potency of drugs are restored. Vaccine prepared
from strong biofilm forming bacteria induces antibodies against Poly-N-
acetyl-β-glucosamine (PNAG) and improves tolerance against S. aureus
infections (Pérez et al., 2009).
Within the microbiota, different species of microorganism can co-
exist and may either show synergism or compete for space and nutri-
tion. The facultative anaerobic C. albicans and anaerobic Clostridium
difficle (C. difficle) are opportunistic pathogens that reside within the
human gut. In the presence of C. albicans, C. difficle can grow in
anaerobic conditions, which would otherwise be toxic. C. difficle in turn
secretes p-cresol that modulates hyphae formation and ultimately in-
hibits biofilm formation in C. albicans (van Leeuwen et al., 2016). Non-
pathogenic microbiota can release antimicrobial molecules in the niche
of pathogenic microbes and block adhesion sites normally used by pa-
thogenic microbes. This type of symbiotic association used by non-pa-
thogenic microbiota increases competition for food or space and also
elicits immune response via IgM, that differs in reactivity to pathogens.
Within the symbiotic environment of the microbiota, a balance in po-
pulation between non-pathogenic and pathogenic microorganisms ex-
ists. Disturbing this balance through antibiotics that selectively target
pathogenic microorganisms may lead to a skewed overpopulation of
non-pathogenic microorganisms, thereby diminishing the chances of
biofilm formation by pathogenic microorganism. The intestinal biofilm
is heavily colonised by wide varieties of bacteria such as Bacteroides
fragilis group, enterobacteria etc., that act as cellular factories for the
breakdown of fatty acid and production of metabolic cofactors (vitamin
B12). These bacteria play an important role in host immunity by
A. Kumar et al.
secreting antimicrobial peptides or competing with pathogenic micro-
organisms for space and nutrition (Macfarlane et al., 2005). Treatment
with antibiotics may sometimes disturb the gut microflora and cause
susceptibility to infection caused by Clostridium spp. (Buffie et al.,
2012). The gut symbiota (probiotics) play an important role in main-
taining microbial composition, metabolism and immunity of gut by
immunomodulating systemic immunity and pH (Singh et al., 2013).
They compete with pathogens for binding sites and neutralize toxins
released by pathogens. Microbiota as probiotics have potentials for use
against biofilms associated with dental plaque, chronic wounds and
urogenital infections (Singh and Hasnain, 2014; Vuotto et al., 2014).
The extracellular matrix proteins, when injected intradermally in
mice, induce humoral immune response and increase Th-2 response.
These mechanisms facilitate phagocytosis of S. aureus and limits bac-
teria, undergoing dispersive stage of biofilm cycle, within the tissue (Gil
et al., 2014). Addition of antibiotics during this treatment regime may
help in an enhanced clearance of bacteria. Although use of whole
protein lysate from biofilm may induce a non-specific immune re-
sponse, recombinant form of specific antigens such as Biofilm asso-
ciated protein (Bap), accumulation associated protein (Aap) and surface
exposed protein induce production of specific antibodies and suppress
bacterial growth (Fattahian et al., 2011; Shahrooei et al., 2012; Yan
et al., 2014).
Antibiotics such as Pyrazinamide shorten treatment duration by
depleting membrane energy and inhibit trans-translation in persister
cells (Zhang et al., 2003; Shi et al., 2011). The ability to target persisters
can significantly improve efforts in eradicating biofilm related infec-
tions. It has been reported that cis-2-decanoic acid reverts persister cells
to drug-susceptible state by improving metabolic activity. A combina-
tion of cis-2-decanoic acid and antimicrobials could thus inhibit bac-
terial viability (Marques et al., 2014). Bacteria develop resistance to
current antibiotics by modifying regulatory pathways that are the tar-
gets of antibiotics, synthesize enzymes for degrading antibiotics, de-
velop efflux mechanisms (Siddiqi et al., 2004), modify cell wall com-
ponents etc. (Wright, 2005). Nanoparticles can be easily modified for
surface charge and ligand binding properties. They exhibit anti-
microbial activity by affecting the cell permeability, generation of re-
active oxygen species or by competitive inhibition of key enzymes.
Since their mode of action is completely different as compared to an-
tibiotics, bacteria have lesser chance of developing resistance against
nanoparticles. Catalytic nanoparticles containing Fe3O4 have shown
promising peroxidase like activity and trigger formation of free radicals
that disrupt biofilm matrix and kill bacteria encapsulated within the
biofilm matrix (Gao et al., 2016). The different infection stages of
mycobacteria confer differential susceptibility towards antibiotics, the
inactive stationary phase being more resilient towards drugs. While
antibiotics are effective in inhibiting the growth of bacteria by targeting
key transcriptional, translational or metabolic pathways, their efficacy
is redundant on non-living polymeric extracellular matrix. Anionic al-
ginates bind to positively charged antibiotics such as fluoroquinolones
or aminoglycosides, impede the penetration of antibiotics and thus re-
quire higher inhibitory concentration of drugs. P. aeruginosa possesses
high affinity siderophores that aid in iron uptake from the host. In CF
patients, free iron is often recorded in sputum and clinical isolates of
lungs (Smith et al., 2013). Use of metal chelators or nanomolar con-
centrations of D-amino acids for dispersal of biofilm prior to treatment
with drugs are emerging as novel strategies for combating biofilm
problem (Banin et al., 2006; Kolodkin-Gal et al., 2010).
Since aggregation of cells constitutes the first step in biofilm for-
mation, strategies to disrupt aggregation by use of quorum sensing in-
hibitors would suppress formation of multicellular structure. There has
been an alarming increase in the number of resistant pathogens due to
indiscriminate use of antibiotics and the inherent ability of micro-
organisms to evolve in order to subvert drug targets, induce immune
skewness or form biofilm resistance. Whereas antibiotics primarily slow
down the growth of microbes, newer strategies combining the use of
6
International Journal of Medical Microbiology xxx (xxxx) xxx–xxx
quorum inhibitors, nanoparticles, biofilm disrupters and antibiotics to
simultaneously target various aspects of microbial physiology can
possibly attenuate their survival.
8. Conclusion
Although a wide array of antibiotics have been developed since the
discovery of Penicillin nearly a century ago, the knockout punch re-
quired for eradicating infections is still eluding researchers. The use of
antibiotics can successfully suppress infection but in majority of cases,
there has been a resurgence of infections. Although survival mechan-
isms employed by microorganisms have been substantively deciphered
and their genomic or proteomic targets identified, rapid evolution of
microorganisms outpace the current profile of chemotherapeutics.
Initial drug resistance endowed by mutations in genes were supposed to
contribute to resilience of infections, but research over the last decade
has shown that biofilms are formed by microorganism across the
spectrum and have been identified in majority of clinical infections.
Cells within biofilms exhibit differential gene expression which pro-
vides heterogeneity to the cohort in space and time. Thus, a certain
genetic pathway active in one cell may be shut off in other cells of the
cohort and subvert the effectiveness of a singular therapeutic strategy.
Besides, biofilm matrix impedes interaction of drugs with microorgan-
isms and provides a protective barrier against immune surveillance.
Within biofilm, a small subpopulation of cells evolve into phenotypi-
cally dormant persister cells that later disseminate from the biofilm,
show enhanced tolerance to drugs and lead to recalcitrance of infec-
tions. The mechanistics of biofilm formation have clearly outlined de-
finitive pathways involving a cycle of attachment, sessile growth, co-
lonization and dissipation. A silver lining to this observation is that
several genes involved in biofilm cycle are common in both pathogenic
as well as non-pathogenic genre and offer an exciting platform for
targeting using inhibitors. It is hypothesised that targeting biofilms
could prove to be the masterstroke required for tackling a wide spec-
trum of microorganisms, particularly those involved in pathogenesis in
humans. A multi-targeted strategy against biofilm formation would
therefore limit resilience of infections commonly observed in hospital
infections.
Funding
SEH and NZE thank the DBT, Ministry of S & T, Government of India
for a Centre of Excellence Grant.
Competing interest
The authors declare that we have no conflict of interests.
Acknowledgements
SEH is a JC Bose National Fellow, Department of Science and
Technology, Government of India and Robert Koch Fellow, Robert Koch
Institute, Berlin, Germany. AK is a recipient of Research Associateship
from the Centre of Excellence Grant (BT/PR12817/COE/34/23/2015)
to SEH and NZE from the Department of Biotechnology (DBT), Ministry
of Science and Technology, Government of India. AA is a recipient of
Research Associateship from Department of Biotechnology, GoI, India.
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