https://revistas.utm.edu.ec/index.php/QhaliKay

Artículo original Enero-Abril 2022;6(1):60-76

https://doi.org/10.33936/qkrcs.v6i1.3601

Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador 60

Biofilms of pathogenic bacteria and emerging antibiofilm strategies

Biopelículas de bacterias patógenas y estrategias emergentes de antibiopelículas

S.A. Aransiola

M.O. Victor-Ekwebelem

M.O. Edward

N.R. Maddela

Abstract

Biofilms act as physical barriers to the immune system and drugs used by the host, resulting in antimicrobial

resistance. Biofilms reduce the chances of eradicating infections and can result in relapses and backsliding

after conventional treatment. Biofilms have a big impact on food safety in the food industry; many foodborne

outbreaks have been linked to pathogenic bacteria that can form a biofilm. Biofilm-associated infections can

cause not only severe symptoms but also serious side effects and even death. The findings of an experimental

study of pathogenic bacteria like Pseudomonas aeruginosa, Salmonella enteritidis, and Staphylococcus aureus

forming biofilms are presented in this article. The process of biofilm formation and its development phases

were displayed with preserved architectonics using light and scanning electron microscopes. The amount of

biofilm formed was influenced by the growth medium as well as the incubation conditions and time. Biofilm-

forming microbes are a common cause of complicated and recurrent diseases, and they are usually linked to

multidrug-resistant bacteria, which account for nearly 80% of all refractory nosocomial infections. Medical

device- and tissue-associated biofilm infections are two types of biofilm infections. Understanding the

pathogenesis and factors that contribute to biofilm formation, as well as the disruption and dispersal

mechanisms of biofilms, will aid in the development of improved anti-biofilm strategies. Overall, this literature

review can serve as a single source of information about microbial biofilm formation and mitigation strategies,

which could be extremely useful to biofilm researchers.

Keywords: biofilm; pathogenic bacteria; infection; anti-biofilm.

Resumen

Las biopelículas actúan como barreras físicas para el sistema inmunitario y fármacos utilizados por el

huésped, lo que genera resistencia a los antimicrobianos. Las biopelículas reducen las posibilidades de

erradicar infecciones y pueden provocar recaídas y recaídas después del tratamiento convencional. Las

biopelículas tienen un gran impacto en la seguridad alimentaria en la industria alimentaria; muchos brotes

de origen alimentario se han relacionado con bacterias patógenas que pueden formar una biopelícula. Las

infecciones asociadas a biopelículas pueden causar no solo síntomas graves, sino también efectos secundarios

graves e incluso la muerte. En este artículo se presentan los hallazgos de un estudio experimental de bacterias

patógenas como Pseudomonas aeruginosa, Salmonella enteritidis y Staphylococcus aureus que forman

biopelículas. El proceso de formación de biopelículas y sus fases de desarrollo se exhibieron con arquitectura

preservada usando microscopios de luz y electrónico de barrido. La cantidad de biopelícula formada estuvo

influenciada por el medio de crecimiento, así como por las condiciones y el tiempo de incubación. Los

microbios formadores de biopelículas son una causa común de enfermedades complicadas y recurrentes, y

por lo general están relacionados con las bacterias multirresistentes, que representan casi el 80 % de todas

las infecciones nosocomiales refractarias. Las infecciones por biopelículas asociadas a dispositivos médicos

y tejidos son dos tipos de infecciones por biopelículas. Comprender la patogenia y los factores que contribuyen

a la formación de biopelículas, así como los mecanismos de interrupción y dispersión de las biopelículas,

ayudará en el desarrollo de estrategias mejoradas contra las biopelículas. En general, esta revisión de la

literatura puede servir como una fuente única de información sobre la formación de biopelículas microbianas

y las estrategias de mitigación, lo que podría ser extremadamente útil para los investigadores de biopelículas.

Palabras clave: biopelícula; bacterias patógenas; infección; antibiopelícula.

*Dirección para correspondencia: raju.maddela@utm.edu.ec

Artículo recibido el 05-07-2021 Artículo aceptado el 26-11-2021 Artículo publicado el 28-02-2022

Fundada 2016 Facultad de Ciencias de la Salud de la Universidad Técnica de Manabí, Ecuador.

Bioresources Development Centre, National Biotechnology Development Agency, Ogbomoso, Nigeria.

Department of Biology/Microbiology/Biotechnology, Alex Ekwueme Federal University, Abakaliki, Ebonyi State, Nigeria.

Bioresources Development Centre, National Biotechnology Development Agency, Ogbomoso, Nigeria.

Universidad Técnica de Manabí, Facultad de Ciencias de la Salud, Departamento de Ciencias Biológicas, Portoviejo, Manabí, Ecuador, raju.maddela@utm.edu.ec,

https://orcid.org/0000-0002-7893-0844

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Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador 61

Introduction

Biofilm is an association of microorganisms that are immovably appended to the biotic or abiotic

surface, encased within an extracellular polymeric substance (EPS) matrix, which might display new

character with reference to metabolic activities, organic phenomenon, rate of growth, and protein

synthesis

1,2

. The EPS are composed of polysaccharides, lipids, proteins, and extracellular DNA

(Table 1) and play a crucial feature within side the pathogenesis of the various microbial infections

Table 1. Chemical composition of biofilm

s/n

Component

Percentage of matrix

Microbial cells

2.5%

DNA and RNA

<1-2%

Polysaccharides

1-2%

Proteins

<1-2% (including enzymes)

Water

Up to 97%

Biofilm production is motivated and influenced by variety of things like surface conditions,

chemical and physical growth factors, cellular structures, and the other challenges. The interaction

between these and other factors determines its fate

4,5

, since biofilms are surrounded by high relative

molecular mass EPS that connect and fix cells, these cells in biofilm can survive harsh growth

conditions

, this takes place thanks to structural and physiological change takes place after cells are

attached to conditioned surfaces, with the produced structural polymeric substances acting as a

barrier

and forestall the doorway of antibiotics and sanitizer agents (Figure 1).

Figure 1. Biofilm formation and structure, adapted from

2,4

It has also been reported that microbial cells within the biofilms are observed to be resistant against

UV, metal toxicity, acid exposure, desiccation, pH gradients, etc.

. Furthermore, biofilm mode of

growth induces microbial resistance to disinfection, which may lead to widespread economic and

health concerns

, for example, a search done on Listeria monocytogenes revealed that its biocide

resistance and cap-potential to cooperate with other species forming heterogeneous communities

allowed this bacterium to survive and struggle within the commercial areas

. In accretion to numerous

physical and chemical tolerances, EPS confers immune resistance to several resident pathogenic

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

62 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

microbes inside biofilms with the help of using inhibiting neutrophil-mediated phagocytosis.

Mosselhy et al.

reported that the eDNA and intercellular adhesins of EPS act as a barrier for the

penetration of a range of antimicrobials. The eDNA present within the EPS chelate human

antimicrobial peptides (AMPs) and reduce the antimicrobial activity of those. Pathogenic

microorganisms can produce biofilm on implanted devices

Many bloodstream infections and tract infections are related to indwelling medical devices which

arise from biofilm consisting of bacteria embedded within an extracellular polysaccharide matrix on

the surface of the catheter

; as an example, Staphylococcus aureus and Staphylococcus epidermidis

are considered two of the foremost important pathogens, and their biofilm regularly causes device-

associated infections

; the biofilm phenotype adapted by these bacteria during device associated

infection facilitates accelerated resistance to antibiotics and host immune defences

The formation of biofilm by microbial pathogens allows them to survive in hosts and causes

chronic infections that achieve persistent inflammation and tissue damage

therefore; formation of

biofilm on medical instruments, human tissues, and organs has an impression on human health and

also the economy. Most bacteria and fungi, like Pseudomonas aeruginosa

, S. epidermidis

Candida albicans

, Acinetobacter baumannii

, Helicobacter pylori

, S. aureus

, L.

monocytogenes

, Vibrio cholerae

, and Salmonella enterica

, are the foremost well-known

pathogenic biofilm formers, these microorganisms form biofilms during a similar manner and share

many common features

22,18,23

Biofilm of pathogenic bacteria

Biofilms are important virulence factors of some pathogenic microorganisms, and a few biofilm

infections seem nearly impossible to eradicate. Most bacteria and fungi, like Pseudomonas

aeruginosa, Staphylococcus epidermidis, Candida albicans, Acinetobacter baumannii, S. aureus,

Helicobacter pylori, L. monocytogenes, Vibrio cholerae and Salmonella enterica are good formers of

biofilms and that they all form biofilms in an exceedingly similar manner with styles of common

featured being shared

13,16,17,18,19,20,21

Biofilms are one in all the foremost important health threats, causing nearly 80% of refractory

nosocomial infections. Biofilm-related infections caused by pathogenic bacteria will be divided into

medical device- and tissue-associated biofilm infections.

The mode of biofilm establishment in several human pathogens, in addition as its drug resistance

mechanism, is well documented and reviewed by different researchers (Figure 2). This figure explains

the shared mechanism of biofilm tolerance under three sections. Physical tolerance: the surplus

production of EPS restricts the penetration and diffusion of antimicrobials; as a result, cells within

the biofilm get longer to become tolerant.

A recent study conducted by Pavlova et al.

, the investigator tries to research the various phases

of biofilm formation and their structure by pathogenic bacteria using the strategy of growing

microorganisms on coverslips in liquid and semi-liquid nutrient media with a 24 to 48 h microbial

cultures within the S-form. Suspension of bacteria at an amount of 10

CFU/ml (according to the

turbidity standard) within the amount of 5 ml was shaken employing a Vortex apparatus and

introduced into Petri dishes with 20 ml of meat-peptone broth. Sterile fat-free coverslips were placed

on sterile slides and immersed in a very liquid nutrient medium in Petri dishes; it absolutely was

incubated in a very thermostat for 24, 48 and 72 h at 37 °C.

The coverslips were removed with tweezers and placed in Petri dishes with paper filters on the

underside so as to preserve natural architectonics, it had been fixed in vivo by vapour of 25 you look

after glutaraldehyde for 2 h. Preparations were coloured by vapour of 4 % solution of OsO

for 3

minutes, then an answer in style of drops was applied on the surface of a paper placed on top of a

dish.

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Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador 63

Figure 2. The final mechanism of biofilm tolerance to numerous antimicrobials. (A) Physical

tolerance: biofilm matrix limits the diffusion of antimicrobials. (B) Passive tolerance: matrix

enzymes inactivate the penetrated antibiotics molecules. (C) Physiological tolerance: persisted cells

within the deeper layer of biofilm induce adaptive SOS (in latin, si opus sit = if there is need)

response and thus become more tolerant

After treatment with osmic acid vapors was done, the biofilms with bacteria turned yellow or

yellowish-brown, these were examined employing a scanning microscope after dehydration with

propylene oxide vapor and gold ion sputtering. During fixation and washing of objects in a very liquid

solution, structure of biofilms undergoes irreversible changes. Bacterial morphology was examined

using an AxioImager A1 optical microscope (Carl Zeiss, Germany) and a TM 4000 scanning

microscope (SEM) (Hitachi, Japan).

The results on the study of the morphology of biofilm formation revealed that S. aureus during

cultivation in an exceedingly liquid medium was seen that on the surfaces of thin coverslips, a

monolayer culture represented by single bacteria within the S-form was initially formed, then clusters

and biofilms were formed after 24-48 h (Figure 3). Similarly, Figure 4 shows that the expansion of

P. aeruginosa was in the middle of formation of a dense biofilm after 48 h. In some areas, single

bacteria developing on the surface are visible; this means possibility of formation of the subsequent

layer of biofilm and thus a multilayer. Presence of pathogenic strains of P. aeruginosa in alginate

causes high resistance to antibacterial and disinfectant drugs. With an outsized increase in SEM,

heteromorphic cells of a spheroplastic, protoplast sort of various sizes with various manifestations of

L-transformation are revealed in fragments of the population under the film and between the clusters

(Figure 5). When S. enteritidis was examined with a scanning microscope, both single adhered

bacteria and populations grouped in clusters were visible. Often channels that are involved within the

metabolism and regulation of oxygen supply were identified inside the formed biofilms. Thus, using

three different microorganisms, we've shown general rules of biofilm formation. Survival strategy of

assorted pathogenic bacteria consists in their evolution by creation of biofilms during which bacteria

are at early stages in vegetative form (S-form) with future transition to heteromorphism with various

manifestations of L-transformation

. L-transformation process is in the course of formation of small

cells with a size of 0.2–0.3 microns- stable and unstable L-forms. The latter compose 20–25 you look

after the entire population. Under favourable conditions, they are reversed to their original state with

preservation of pathogenicity and other biological features.

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

64 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

Biofilm structure can have different composition, counting on the kind of pathogenic bacteria

having a particular structure of the semipermeable membrane and containing peptides, lipids and

other components.

Figure 3. Formation of S. aureus clusters. Inside the clusters, there is a biofilm with bacteria. SEM

× 2500

Figure 4. Formation of a P. aeruginosa biofilm; on the surface, there are single bacteria emerging

from the film. Optical microscopy

Under certain conditions, a multilayer biofilm may develop. This process is related to emergence

of single bacteria on the surface of the upper layer. Then, filamentous structures are formed by

bacteria, creating clusters with an exopolysaccharide matrix during which bacteria multiply and this

contributes to the formation of subsequent biofilm layer (Figure 6). Study of biofilms of gram-positive

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Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador 65

and gram-negative bacteria indicates a sequence of phases of development and existence of bacterial

populations.

Figure 5. Fragment of formation of S. enteritidis clusters of spheroplast and protoplast cells

included in the biofilm. SEM, ×5000

Figure 6. Multi-layer biofilm S. enteritidis 72 hours after cultivation. Single cells are visible on

the upper layer

Biofilms have a significant impact on food safety in the food industry, according to a study

conducted by Joanna et al.

, which found that pathogenic bacteria such as L. monocytogenes, S.

aureus, Escherichia coli, and Bacillus cereus are dominant in retail foods and are the leading cause

of food infection and disease due to their strong biofilm formation and high metabolic activity.

Drug resistance versus biofilm formation

Drug resistance or resistance to antimicrobial agents may be a critical problem and a significant

threat to animal and health. it’s imperative to grasp how bacteria develop resistance to antibiotics.

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

66 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

Much of our understanding of the mechanisms of antibiotic action and resistance comes from

experiments during which bacteria are grown in liquid culture before being exposed to antibiotics.

Yet, most bacteria in nature exist in biofilms, aggregated communities of cells encased during a

matrix

. Biofilms represent a fundamentally different mode of life to planktonic cultures and studies

have demonstrated extreme changes in gene and protein expression profiles from the identical strains

when grown in liquid or as a biofilm

. Many infections include a biofilm component that produces

the infection are difficult to treat; common examples include infections on prosthetic or indwelling

devices. Biofilms are typically more tolerant to antibiotics, compared to the corresponding strain in

liquid culture. One theory explaining the resistance to antibiotics of biofilms is that cells within a

biofilm are metabolically inactive and a high proportion are dormant ‘persisted’ cells. In these

dormant subpopulations, characterised by arrested macromolecular syntheses, the cellular targets that

the antibiotics poison are often not essential, thus impeding bactericidal activity

. Despite this

reduced rate, biofilms are shown to be able to adapt rapidly to changing conditions, and rapid selection

of mutants with improved biofilm fitness is observed when bacteria are introduced to a replacement

niche

Biofilms are groups of microorganisms attached to biotic or abiotic surfaces and surrounded by a

matrix composed of an EPS (Figure 7)

. Biofilms exist in various infections and are demonstrated to

play a crucial role in human diseases, they act as physical barriers against drugs and host immune

responses, resulting in resistance to antimicrobial treatment and clearly reduce the likelihood of

eradicating infections which cause relapses after the standard appropriate treatment.

Figure 7. The structure of the extracellular polymeric substance

. The onset of biofilm-related

infections can increase not only severe symptoms but also mortality

. Although more studies are

that specialize in strategies to eliminate microbial biofilms, but it's better to know the roles of

biofilms in infections and its mechanisms to drug resistance for this can assist in proffering solution

like latest promising antibiofilm strategies (QS – Quorum sensing).

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Biofilm formation

Formation of biofilm mainly involves three stages (Figure 8 and 9); the primary stage is that the

adhesion stage; cells attach to a surface; within the second stage (sessile growth stage), micro-colonies

are formed thanks to the assemblage of those cells. The adhesion and sessile stages of growth are

reversible and therefore the cells can cluster loosely but can detach and return to a planktonic state

thereafter, the attached cells secrete EPS, which incorporates extracellular DNA (eDNA),

polysaccharides and proteins (Figure 8) which developed into a biofilm within the third stage. This

stage is irreversible, because the cells are attached within a thick and stable complex bio-molecular

layer

. After a biofilm is totally developed, its dispersion or disassembly occurs via both active and

mechanical processes, these processes occur within the fourth stage (dispersal stage). The cells within

the biofilm secrete not only cell–cell-adhesive matrix components but also disruptive factors,

including proteases, nucleases, phenol-soluble modulins, and regulators

. These disruptive factors

can even promote biofilm detachment. During the method of detachment, biofilms can shed

individual cells and slough off pieces into the bloodstream and also the surrounding tissues, which

are related to many acute and chronic infections

. Cells with different phenotypes and genotypes co-

express individual metabolic pathways, stress responses, and other distinct biological properties

within the biofilms. A number of these cells alter extracellular polysaccharide and organelle

production and even cell morphology after they sense growth within the biofilm community. DNA

transfer and genetic recombination between the multiple microbial species within a biofilm occur

without direct cell–cell contact through the extracellular matrix and during this manner antibiotic

resistance genes may be transferred

3,35

Figure 8. Schematic of biofilm formation. (A) The structure of the extracellular polymeric

substance. (B) The stages of biofilms. (C) The catheter-associated biofilm

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

68 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

Quorum sensing (QS) is a cell-to-cell communication process that controls and facilitates biofilm

formation in many bacterial and fungal species, resulting in antibiotic resistance and the production

of virulence factors

36,37,5,28

. QS is widely recognized as essential for genetic regulation and population

dynamics and plays vital roles in biofilm development. In order to optimize metabolic production,

microorganisms use QS to regulate population density

. By inhibiting the synthesis of matrix

compounds or the degradation of the matrix in a coordinated manner

, QS controls not only the

maturation but also the disassembly of the biofilm community.

Figure 9. The stages of biofilm formation and role of QS

Bacteria attach to matrix-forming proteins in humans via peptidoglycan structure, either covalently

or non-covalently. With the attachment and aggregation of a sufficient number of cells, the EPS

matrix is formed, and the attachment is now resistant to external repulsive forces. As the biofilm

matures, the cells within the bulk structure begin to communicate and secrete specialized proteins and

DNA, and some of these proteins and DNA are involved in the formation of the efflux pump. Finally,

the dispersion of free planktonic cells from the formed biofilm promotes the formation of new

biofilms in the periphery.

Mechanisms of biofilm resistance to antimicrobial agents

The mode of growth of biofilm gives protection against many biocides and antibiotics; thus,

biofilms are hard to regulate and ultimately eradicate. It’s been indicated that microorganisms re-

suspended from biofilms are distinctly more resistant than planktonic cells, while the cells inside

biofilms are more resistant than those re-suspended from biofilms. Biofilm cells are a minimum of

many times more immune to antibacterial agents than planktonic cells (up to 1,000-fold increase)

Biofilms protect cells from desiccation, chemical perturbation, invasion by other bacteria, and killing

by immune cells by acting as shelters or physical barriers

There are multiple mechanisms by which biofilm cells create increased resistance to antibiotics,

and these mechanisms are distinct from those in planktonic cells (Figure 10). Impeded antibiotic

penetration into biofilms was initially proposed to be responsible; however, penetration by some

antimicrobial agents, like ciprofloxacin and fluconazole, throughout biofilms doesn't decrease

. It’s

now well-known that the matrix mesh size is far larger than the scale of antibiotic molecules

. The

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penetration of antimicrobials into a biofilm depends on the thickness of the biofilm, the reactivity and

diffusion of the agent within the biofilm, the sorption of the biofilm, and therefore the dose

concentration of the agent

. the assembly of an exopolysaccharide matrix reduces the activity of

some antibiotics, like fluconazole, in C. albicans biofilms, the eDNA is considered one in every of

the foremost important contributors to the resistance of biofilms to antimicrobial agents, by reducing

the activity of antibiotic through creating cation-limited conditions, inducing modification of

lipopolysaccharide (LPS), and impairing the uptake of antibiotics, like aminoglycosides

Figure 10. Mechanisms of biofilms that are resistant to antibiotics therapy

It is widely accepted that the status of the cells within biofilms is related to their sensitivity to

antimicrobials. The upper osmolarity conditions, greater oxygen limitations, higher metal ion

concentrations, and lower pH levels within a biofilm are confirmed to be chargeable for the expression

of some genes and help determine a number of the phenotypes of biofilm cells

. The concentration

of oxygen within a biofilm is higher at the surface and lower at the underside and also the centre with

cells having high level of metabolic activity located at the surface of the biofilm while those with a

coffee level of metabolic activity and slow growth are found within the canter. Metabolically active

cells are able to sense environmental changes and actively reply to the presence of antimicrobial

stress. However, majority of the cells inside biofilms are in a very dormant state and within the

stationary phase, which implies that these cells are metabolically inactive and not growing. Cells

within the stationary phase within a biofilm do not grow and respire and are more tolerant to

antimicrobials

. Changed nutrient environments and inhibition of growth within the biofilm result in

increased drug resistance within biofilms.

Intrinsic mechanisms of resistance are present in biofilms, but many studies have indicated that the

synergy of acquired and adaptive mechanisms contributes to antibiotic resistance in biofilms

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

70 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

Genetic adaptation within biofilms helps cells adapt to their surroundings and increases their

antibiotic resistance. Changes within the outer membrane proteins of the cells within biofilms

contribute to antibiotic resistance via the expression of multidrug resistance genes. Some antibiotics

can induce resistance-related enzyme expression within the cells within biofilms. As an example,

high-level imipenem resistance is said to increased beta-lactamase expression induced by imipenem

in P. aeruginosa biofilms. Piperacillin also can induce beta-lactamase expression in biofilms;

however, the increased beta-lactamase expression isn't as high because the imipenem level

Biofilm persistence in chronic infections is primarily due to a combination of increased beta-

lactamase expression and other protective biofilm growth mode properties

. Drug resistance is

exacerbated by changes in multidrug efflux pump activity in biofilms. The biofilm cells' activated

efflux pumps have gotten the most attention

. Persister cells, which make up the majority of cells in

biofilm communities when they are stationary, are dormant microbial subpopulations that are

phenotypic multidrug-tolerant variants rather than genetic variants

. The acquisition of multidrug

resistance genes by horizontal transfer, which contributes to the evolution of biofilm cells, is another

mechanism of antibiotic resistance in biofilm cells. QS plays a critical role in horizontal cell-to-cell

communication within biofilms. Evidence suggests that biofilms have evolved these mechanisms as

a general stress response that causes microorganisms in the biofilm to react to environmental

changes

47,34

. Novel biofilm-fighting strategies that target these mechanisms must be developed.

Emerging anti-biofilm strategies

Biofilms can be removed by three different mechanisms, such as desorption, detachment and

dispersal

. Reversal phenomenon of bacterial attachment is called as desorption, for instance, leaving

of surface attached cells from the substratum and subsequent entry into bulk fluid. Detachment can

be described as passive dislocation of biofilm embedded cells, and detachment usually happened

when biofilm structure is disrupted by external forces. Whereas in biofilm dispersion, biofilm

embedded cells are actively disseminated from the biofilm, which usually happened when any

changes occurred in the environment. Recently, several biofilm mitigation strategies have been

emerged to overcome the biofilm-based problems in different areas, and thus the biofilm control in

different environmental media is one of the active research areas. Biofilms were found to be

controlled successfully by using different strategies, such as ionic lipids

, electrochemical

treatment

, photocatalytic graphitic carbon nitride-chitosan composites

, phenol derivatives

bioactive glycolipids

, ultrasound treatment

, magnetic nanoparticles

, quorum quenching

combination of high intensity ultrasound and chlorine dioxide

, combined ciprofloxacin and

azithromycin free and nano formulations

, novel phage ZPAH7

, tea polyphenols

, etc. However,

there is no single universal method of biofilm control in different environmental media. In addition,

most of the above-mentioned strategies have been tested against one given environment, and most

with model strains. Therefore, in depth insights are necessary to know the complete scenario of

biofilm formation, such as EPS functional groups

, quorum sensing

and quorum quenching

mixed culture behaviour

In the recent past, there is much attention on the control of P. aeruginosa biofilms as this is one of

the potential multi-drug resistance human pathogen. In this direction, several anti-biofilm agents have

been emerged against P. aeruginosa, such as phytochemicals, nanoparticles, metal chelators,

enzymes, antimicrobial peptides, antibodies, bacteriophage cocktails, plant products (e.g.

isothiocyanates and organosulfur compounds)

. However, use of combination of agents giving better

results, for instance, 10 nm AgNPs (silver nanoparticles) and aztreonam were found to decrease 98%

of biomass and 50% reduction in the thickness biofilm of P. aeruginosa PAO166. Similarly, AgNPs

along with colistin displayed enhanced antibiofilm properties than colistin alone. Nonetheless, for the

establishment of a sustainable antibiofilm strategy for the control of P. aeruginosa infections, more

in vitro and in vivo studies are necessary

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Biofilm mitigation is a becoming a challenging, which is attributed to the complexity in the biofilm

formation potential of different environmental isolates. Especially in industries, biofilm mitigation is

of great importance, as biofilms cause significant economic loss by various reasons such as corrosion

followed by deterioration of equipment, increased fluid resistance, etc

. Also, biofilm contamination

in the food industries can show negative effects on the quality of food products. Similar effects are

also appeared in the paper industry, where biofilm formation affect the final quality of the paper

Another great concern associated with the biofilm formation is human health and innumerable species

of bacteria and fungi capable of producing biofilms and subsequently cause potential health disorders

in the humans. It is widely known fact that control of bacteria in its sessile growth phase is much

harder than bacteria in a free-floating state. Thus nowadays, several mathematical models have been

emerged

66,67

to know the key roles of QS, multi-species interactions, antimicrobial resistance,

mechanical properties of extracellular matrix; such insights are expected to provide in depth

understanding of what properties of biofilms support the bacteria embedded with in a competitive

edge against treatment. Mathematical models are also available to strengthen the idea of how to

prevent the unwanted biofilms in different environmental facilities such as food and water

. It is also

important to note that the interaction of biofilm cells with surface is vary depending on the

architecture of the biofilm. In monolayer biofilms, cell-to-surface interactions are much important

than interactions between the constituent cells (cell-to-cell)

. In fact, bacterial external structures

(e.g. flagellum and pilus) are the key structures accelerate the rate of formation of the monolayer

biofilm. But bacterial cells tend to produce multi-layered biofilms when dual interactions are

involved, such as cell-surface and cell-cell interactions

. In addition, during the formation of

multilayer biofilms, there is a masking and neutralization of repulsive forces of negative surface

charges by means of one of several mechanisms such as mutations, down-regulation of the O antigen

encoding gens, additional of divalent cations, synthesis of EPS

. These types of mechanistic insights

are very helpful in the design or search of a sustainable biofilm mitigation strategy.

Quorum sensing inhibitors (QSI) are another choice of biofilm mitigation agents. They found to be

effective in the control of both growing biofilm and established biofilms

. Cyclic dipeptides (e.g.

cyclo (L-Pro-L-Val)) and others (e.g. cyclo (L-4-iodo-Phe-L-Pro), cyclo (L-4-chloro-Phe-L-Pro)) are

potential QSIs, and effectively mitigate the LuxR-based based QS activities such as biofilms and

luminescence in modelled bacteria, e.g. P. aeruginosa

and Vibrio fischeri

, respectively.

Importantly, QSIs have a capacity to inhibit biofilms of not only the bacteria but also fungi; for

example, biofilms of C. albicans have been mitigated by several types of QSIs like farnesol, farnesoic

acid, tyrosol, tryptophol, and phenylethyl alcohol

. Farnesol is very effective in controlling the later

stages of biofilm development in C. albicans, where germ tube formation and hyphal-inducing

conditions are badly affected by farnesol

. Like farnesol, sesquiterpene alcohol farnesol is also

known to block the yeast to hyphal switch and biofilm formation in C. albicans

Conclusions

Biofilms are sessile microbial cell communities embedded in an EPS, forming a matrix which

adhere to surfaces of medical implants and wounds, to periprosthetic tissue and enables cell to cell

adhesion and aggregation, leading to the formation of flocs. The matrix consists of bacterial secreted

polymers, mostly, exopolysaccharides, lipids, proteins, and extracellular deoxyribonucleic acid (e-

DNA); which aids the intricate three-dimensional (3D) structure and excessive resistance or tolerance

against drug in bacteria. The e-DNA could prompt the expression of resistance genes and the

horizontal gene transfer between bacterial cells within biofilms.

Conflicts of interest

The author declare that there is no conflict of interest.

Biofilms of Pathogenic Bacteria and Emerging Antibiofilm Strategies

Aransiola, Victor-Ekwebelem, Maddela

72 Facultad de Ciencias de la Salud. Universidad Técnica de Manabí. Portoviejo, Ecuador

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