MICROBIAL BIOFILMS AND THEIR IMPACT ON HUMAN HEALTH AND

INDUSTRY

BY

SUNDAY HAPPY
AST/2382280155

A SEMINAR PAPER PRESENTED TO THE DEPARTMENT BIOLOGICAL SCIENCE

LABORATORY TECHNOLOGY, SCHOOL OF APPLIED SCIENCE AND
TECHNOLOGY, AUCHI POLYTECHNIC AUCHI

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

HIGHER NATIONAL DIPLOMA (HND) IN (BIOLOGY/MICROBIOLOGY OPTION)

FEBRUARY, 2024

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 OUTLINE

 Abstract
 Introduction
 Microbial Biofilms
 Biofilm Development

 Attachment: a surface-sensing step

 Growth or microcolony formation
 Bacteria That Forms Biofilms
 Properties of Microbial Biofilms

 Habitats of Biofilms
 Biofilms and Human Health
 Infections Attributed to Biofilms
 Biofilm In industry
 Food industry
 Methods to Control Biofilms

 References


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 Abstract
The term biofilm is used to denote a polymer-encased community of microbes which
accumulates at a surface. Biofilms are responsible for a number of diseases of man and, because
of the intrinsic resistance of these structures to antibiotics and host defence systems, such
diseases are very difficult to treat effectively. Example of such bacteria are E-coli, strep,
staph.psudom. During the development of Biofilms, some steps must be met such as Bacterial
landing of the surface , it tend to attach , followed by growth, then maturation and it dispenses.
The application of new microscopic and molecular techniquesto biofilms has revolutionised our
understanding of their structure, composition, organisation and activities. This review will
describe the Impact of biofilms human health and industry and will outline our new millennial
view of these complex and fascinating bacterial communities

Keywords: Microbial Biofilms, Human Health, Industry

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Introduction

Microorganisms can live in free form or in association of different or same species, called
biofilm. Biofilms are an ordered and arranged group of microorganisms living within an
extracellular polymeric substance (EPS) matrix produced by them and are adhered to each other
on living or non-living surfaces and show variations in terms of growth rate and gene expression
when compared to their planktonic form (Gupta, et al., 2016). To develop a relationship with the
host, to show resistance towards hostile external conditions, and to cope with the known
antibiotics and other environmental cues, the microorganisms have evolved to form a protective
cover around themselves (Castiblanco & Sunding, 2019).

Biofilm formation contributes towards the development of antibiotic resistance and the formation
of persistent cells which are responsible for the unmanageable persistence of microbial
infections. Biofilms have various pathological manifestations and exist almost everywhere,
inhabiting medical implants, living tissues, water channels, pipes, hospital floors, food
processing units, and other biotic and abiotic surfaces. Changes in phenotype and gene
expressions accompanied by the resistance to known antibiotics, metabolic activity and growth
rate reduction, and production of virulence-associated factors are some features of biofilm-
associated microorganisms (Sharma, et al., 2016).

As per the reports of the National Institutes of Health (NIH), about 65% and 80% of microbial
and chronic infections, respectively, are caused by microbial biofilms, infecting both tissues and
medically implanted devices. Reference breast implants, ventricular shunts, tissue fillers,
ventricular-assisted devices, contact lenses, catheters, joint prostheses, urinary catheters,
orthopedic implants, pacemakers, mechanical heart valves, defibrillator, vascular grafts,
endotracheal tubes, voice prostheses, etc. are some examples of medically implanted devices
often infected by microbial biofilms. Some of the tissue-related infections caused by microbial
biofilms include periodontitis, osteomyelitis, lung infection in cystic fibrosis, endocarditis, dental
plaque, chronic tonsillitis, chronic laryngitis, chronic wounds, and biliary and urinary tract
infections (Rather, et. al., 2021).

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Furthermore, different sectors of the food industry, viz. poultry, dairy, ready-to-eat, and
aquaculture. They are severely affected by biofilm-producing microorganisms resulting in food
spoilage, disease outbreaks, and deaths. So, keeping in view the prevalence of biofilm-associated
microorganisms and inefficiency of current antibiotics, the situation requires a transition towards
the formation of non-toxic and potent antibiofilm agents targeting signaling pathways regulating
quorum sensing (QS), EPS synthesis, biofilm-related genes, microbial motility, adhesion,
dispersion, and many more (Roy, et al., 2018). The recent novel antibiofilm approaches include
the use of ultrashort antimicrobial peptide nanoparticles, host defense peptides (HDPs), surface-
active organosilane biocide-Goldshield (GS5), biofilm-specific peptides, smart antibiofilm
surfaces, nanoelements (NEs) , and poly(ether urethane) (PEU) films for disposal of antibiofilm
agents like gallium (Ga) or zinc (Zn) (Ma, et al., 2017).

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Microbial Biofilms

Microbial biofilms are complex microbial communities encased in extracellular polymeric

substances. To put it another way, A biofilm is a community of microorganisms, such as
bacteria, for example E-coli, Staph. Spp, Strapp spp, Pseudomonas aeruginosa that are capable
of living and reproducing as a collective entity known as a colony. Biofilms can also be defined
as living biomass that possess a sophisticated social structure that personnel involved in this
field are still attempting to decipher. The structure of biofilm serves both to shield and enable the
expansion of the colony (Gupta, et al., 2016).

Traditionally, bacteriologists have studied most aspects of bacterial structure and behaviour
using cells that have been grown suspended in a liquid medium. Organisms grown in this way
are described as being “planktonic”. However, it is increasingly being realised that, in their
natural habitat, most bacteria grow attached to surfaces i.e. they are “sessile”1. Furthermore, the
growth of many sessile bacteria results in the formation of large aggregates and these are known
as “biofilms” (Watnick, & Kolter, 2020).

In man, the surfaces available for attachment are many and varied and all surfaces exposed to the
external environment (i.e. the skin, teeth, respiratory and intestinal mucosa etc.) support
populations of sessile bacteria. However, as the majority of such surfaces are continually being
shed (along with the bacteria attached to them) and renewed, the opportunities for biofilm
formation are more limited. The only natural, non-shedding surfaces in man exposed to the
environment are those provided by the teeth and here we find excellent examples of true biofilms
(as opposed to sessile bacteria) – dental plaques (Newman & Wilson, 2019).

Biofilm Development

Biofilm formation is a multi-step and complex process that involves the transition of bacteria
from free-swimming planktonic form to biofilm-making sessile form. The whole process of
formation is influenced by external conditions like temperature, pH, gravitational forces,
hydrodynamic forces, Brownian movements, nature of the inhabiting surfaces, quorum sensing,
secondary messengers, and other signaling molecules as well. The formation of Biofilm can be
divided into four major steps (Chandki et. al., 2019).

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 Attachment: a surface-sensing step
The process of biofilm formation is triggered with the adherence of planktonic
microorganisms to surfaces and, thus, considered as an important stage to develop the
free-flowing microorganisms into an assembled community structure (Haggag, 2016).
During the initial stage of biofilm formation, microorganisms are loosely and reversibly
attached to surfaces and this stage is characterized by the presence of polarly attached
microorganisms to the surfaces. Thereafter, microorganisms change the orientation to lay
flat on the surfaces and go for irreversible attachment which develops resistance to many
physical factors hindering biofilm formation (Banerjee, 2015).
 Growth or microcolony formation
Soon after the successful adhesion of microorganisms to the surfaces, the adhered
microorganisms start multiplication and aggregation within self-produced EPS leading to
the microcolony formation in presence of a high concentration of c-di-GMP. Flagella and
type IV pili-mediated motilities are important for interactions between microorganisms
and surfaces, and cell–cell aggregations to form microcolonies, respectively (Rabin, et
al., 2015).
 Maturation
Stage four of biofilm formation is maturation where the attached cells mature and
develop further. Maturation is enabled by the secretion of signaling factors by the
attached bacterial cells resulting in the expression of biofilm-specific genes. Signaling
factors help alter gene regulation to enhance bacterial virulence. The process begins with
the release of EPS from the cells, which stabilizes the biofilm structure and shields it
from antimicrobial agents (Gupta, et al., 2016; Veerachamy, et al., 2014). For example,
P. aeruginosa generates unique saccharides (alginate, Pel, and Psl) throughout maturation
that offer biofilm stability.

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 Dispersion

Finally, matured biofilm ruptures actively (motility and EPS degradation–dependent

dispersion) or passively (physical factors like liquid flow-dependent dispersion) to
disperse the microorganisms to start a new cycle of biofilm formation(Habash, & Reid,
2019). Some factors which are mainly responsible for the dispersion of matured biofilm
include outgrown population, intense competition, lack of nutrients, enzyme action that
causes alginate digestion in Pseudomonas spp., and variation in environmental conditions
like temperature, oxygen deficiency, and metabolite accumulation as well as up
regulation of genes responsible for cell motility and EPS degradation, and down
regulation of genes important for polysaccharide and fimbriae synthesis (McDougald, et
al., 2017).

Fig. 1─Biofilm formation

Source: (Banerjee, et al., 2015)

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Bacteria That Forms Biofilms

Most bacteria, given the right conditions, can grow as a biofilm. However, certain species appear
to have a predilection to form biofilms and examples of these are given in Table 1.

Table 1: Examples of organisms that frequently form biofilms

Organism Site of biofilm formation

Staphylococcus aureus Implantable medical devices
Staphylococcus epidermidis and other Implantable medical devices
coagulase-negative staphylococci
Pseudomonas aeruginosa Lungs of cystic fibrosis patients
Escherichia coli and other enterobacteria Urinary catheters
Escherichia coli Intestinal tract
Streptococcus spp Teeth
Actinomyces spp. Teeth
Lactobacillus spp Vagina, teeth

Sources: (Stefani & Agodi, 2020).

Most of these species are members of the normal microflora of man and form biofilms at sites
where they are found naturally. Hence, streptococci, cariogenic bacteria and
periodontopathogenic bacteria form biofilms on the surfaces of teeth, while lactobacilli form
biofilms in the vagina. Staphylococci, which are members of the normal microflora of the skin,
often form biofilms on implantable medical devices which penetrate the skin (e.g. central venous
catheters) or they gain access to totally-implanted devices such as hip, knee and other joint
prostheses. Escherichia coli can form biofilms on urinary catheters (as can a variety of other
members of the intestinal microflora) and also in the intestinal tract. Of the organisms listed in
Table 1, Pseudomonas aeruginosa is exceptional in that it is not a member of the normal
microflora but is an environmental species which is a notorious opportunistic pathogen of
individuals whose defence systems are impaired in some way. Hence, it often causes infections
of burns and wounds and is a major problem for immunocompromised individuals.

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Properties of Microbial Biofilms

Biofilms are usually found on solid substrates submerged in or exposed to an aqueous solution,
although they can form as floating mats on liquid surfaces and also on the surface of leaves,
particularly in high humidity climates. Given sufficient resources for growth, a biofilm will
quickly grow to be macroscopic (visible to the naked eye). Biofilms can contain many different
types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performs
specialized metabolic functions. However, some organisms will form single-species films under
certain conditions. The social structure (cooperation/competition) within a biofilm depends
highly on the different species present (Toyofuku, et al., 2016).

Habitats of Biofilms

Biofilms will form on virtually every non-shedding surface in non-sterile aqueous or humid
environments. Biofilms can grow in the most extreme environments: from, for example, the
extremely hot, briny waters of hot springs ranging from very acidic to very alkaline, to frozen
glaciers (Andersen et al., 2017).

Biofilms can be found on rocks and pebbles at the bottoms of most streams or rivers and often
form on the surfaces of stagnant pools of water. Biofilms are important components of food
chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish
feed. Biofilms are found on the surface of and inside plants. They can either contribute to crop
disease or, as in the case of nitrogen-fixing rhizobia on root nodules, exist symbiotically with the
plant. Examples of crop diseases related to biofilms include citrus canker, Pierce's disease of
grapes, and bacterial spot of plants such as peppers and tomatoes (Chandki, et al., 2019).

Biofilms and Human Health

While much emphasis is placed on the adverse effects of biofilms and the difficulty in treating
diseases which they cause , it must be emphasised that some biofilms have a protective role.
Hence, biofilms (composed mainly of lactobacilli) in the vagina prevent colonisation by
exogenous pathogens (a phenomenon known as “colonisation resistance”) and, indeed, their
presence is usually synonymous with vaginal health. The ability of the biofilm to prevent
colonisation by pathogens is attributable to the production of acids, bacteriocins, hydrogen
peroxide, and biosurfactants (Prigent, & Lejeune, 2019)

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Disruption, or disappearance of this protective biofilm, is used as an indication of the presence of
vaginal pathogens such as Gardnerella vaginalis or other anaerobes 15. Indeed, re-colonisation of
the vagina with lactobacilli is advocated by many as an appropriate means of treating vaginal
infections. Dental plaque, the biofilm that forms on the surface of teeth, also protects against
colonisation by exogenous pathogens. While this biofilm consists mainly of streptococci and
Actinomyces spp., many other species may be present and, under certain conditions (to be
discussed later), such species can proliferate resulting in a biofilm that is not compatible with
health. These biofilms can induce diseases such as caries, gingivitis and periodontitis which are
among the most common infections of man (Donelli, et al., 2014).

Apart from oral infections, human diseases due to biofilms are usually associated with the
presence of some implantable medical device (e.g. catheters, joint prostheses) or are the
consequence of some impairment of the host defence systems e.g. lung infections in cystic
fibrosis patients (Costerton, et al., 2019)

In human environment, biofilms can grow in showers very easily since they provide a moist and
warm environment for them to thrive. They can form inside water and sewage pipes and cause
clogging and corrosion. On floors and counters, they can make sanitation difficult in food
preparation areas. In soil, they can cause bioclogging. In cooling- or heating-water systems, they
are known to reduce heat transfer (Kim et al., 2016).

Infections Attributed to Biofilms

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by
one estimate 80% of all infections. Infectious processes in which biofilms have been implicated
include common problems such as bacterial vaginosis, urinary tract infections, catheter
infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses,
and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and
infections of permanent indwelling devices such as joint prostheses, heart valves, and
intervertebral disc. The first visual evidence of a biofilm was recorded after spine surgery. It was
found that in the absence of clinical presentation of infection, impregnated bacteria could form a
biofilm around an implant, and this biofilm can remain undetected via contemporary diagnostic
methods, including swabbing. Implant biofilm is frequently present in "aseptic" pseudarthrosis

8
cases. Furthermore, it has been noted that bacterial biofilms may impair cutaneous wound
healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds
(Vyas & Wong, 2016).

It has been shown that biofilms are present on the removed tissue of 80% of patients undergoing
surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of
cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell
morphology. Biofilms were also found on samples from two of 10 healthy controls mentioned.
The species of bacteria from intraoperative cultures did not correspond to the bacteria species in
the biofilm on the respective patient's tissue. In other words, the cultures were negative though
the bacteria were present. New staining techniques are being developed to differentiate bacterial
cells growing in living animals, e.g. from tissues with allergy-inflammations (Leevy et al.,
2016).

 Pseudomonas aeruginosa

P. aeruginosa represents a commonly used biofilm model organism since it is involved in

different types of biofilm-associated chronic infections. Examples of such infections include
chronic wounds, chronic otitis media, chronic prostatitis and chronic lung infections in cystic
fibrosis (CF) patients. About 80% of CF patients have chronic lung infection, caused mainly by
P. aeruginosa growing in a non-surface attached biofilms surround by PMN. The infection
remains present despite aggressive antibiotic therapy and is a common cause of death in CF
patients due to constant inflammatory damage to the lungs. In patients with CF, one therapy for
treating early biofilm development is to employ DNase to structurally weaken the biofilm
(Toyofuku et al., (2016).

 Streptococcus pneumoniae

Streptococcus pneumoniae is the main cause of community-acquired pneumonia and meningitis

in children and the elderly, and of sepsis in HIV-infected persons. When S. pneumoniae grows in
biofilms, genes are specifically expressed that respond to oxidative stress and induce competence
(Mazaheri et al., 2015), Formation of a biofilm depends on competence stimulating peptide

9
(CSP). CSP also functions as a quorum-sensing peptide. It not only induces biofilm formation,
but also increases virulence in pneumonia and meningitis.

It has been proposed that competence development and biofilm formation is an adaptation of S.
pneumoniae to survive the defenses of the host. In particular, the host's polymorphonuclear
leukocytes produce an oxidative burst to defend against the invading bacteria, and this response
can kill bacteria by damaging their DNA (Matsumoto, 2018).

 Escherichia coli

Escherichia coli biofilms are responsible for many intestinal infectious diseases. The
Extraintestinal group of E. coli (ExPEC) is the dominant bacterial group that attacks the urinary
system, which leads to urinary tract infections. The biofilm formation of these pathogenic E. coli
is hard to eradicate due to the complexity of its aggregation structure, and it has a significant
contribution to developing aggressive medical complications, increase in hospitalization rate, and
cost of treatment. The development of E. coli biofilm is a common leading cause of urinary tract
infections (UTI) in hospitals through its contribution to developing medical device-associated
infections. Catheter-associated urinary tract infections (CAUTI) represent the most common
hospital-acquired infection due to the formation of the pathogenic E. coli biofilm inside the
catheters (Reisner, et al., 2014).

 Staphylococcus aureus
Staphylococcus aureus pathogen can attack skin and lungs, leading to skin infection and
pneumonia. Moreover, the biofilm infections network of S. aureus plays a critical role in
preventing immune cells, such as macrophages from eliminating and destroying bacterial cells.
Furthermore, biofilm formation by bacteria, such as S. aureus, not only develops resistance
against antibiotic medication but also develop internal resistance toward antimicrobial peptides
(AMPs), leading to preventing the inhibition of the pathogen and maintaining its survival (Craft
et al., 2019)

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Table 2: Diseases caused by biofilms (AGNB = aerobic Gram-negative bacilli; CNS =
coagulase-negative staphylococci)

Source: (Allison, et al., 2020)

Biofilm In industry

Biofilms can also be harnessed for constructive purposes. For example, many sewage treatment
plants include a secondary treatment stage in which waste water passes over biofilms grown on
filters, which extract and digest organic compounds. In such biofilms, bacteria are mainly
responsible for removal of organic matter (BOD), while protozoa and rotifers are mainly
responsible for removal of suspended solids (SS), including pathogens and other
microorganisms. Slow sand filters rely on biofilm development in the same way to filter surface
water from lake, spring or river sources for drinking purposes. What is regarded as clean water is
effectively a waste material to these microcellular organisms. Biofilms can help eliminate
petroleum oil from contaminated oceans or marine systems. The oil is eliminated by the
hydrocarbon-degrading activities of communities of hydrocarbonoclastic bacteria (HCB).

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Biofilms are used in microbial fuel cells (MFCs) to generate electricity from a variety of starting
materials, including complex organic waste and renewable biomass. Biofilms are also relevant
for the improvement of metal dissolution in bioleaching industry, and aggregation of
microplastics pollutants for convenient removal from the environment (Chan, et al., 2022).

Food industry

Biofilms have become problematic in several food industries due to the ability to form on plants
and during industrial processes. Bacteria can survive long periods of time in water, animal
manure, and soil, causing biofilm formation on plants or in the processing equipment. The
buildup of biofilms can affect the heat flow across a surface and increase surface corrosion and
frictional resistance of fluids. These can lead to a loss of energy in a system and overall loss of
products. Along with economic problems, biofilm formation on food poses a health risk to
consumers due to the ability to make the food more resistant to disinfectants As a result, from
1996 to 2010 the Centers for Disease Control and Prevention estimated 48 million foodborne
illnesses per year. Biofilms have been connected to about 80% of bacterial infections in the
United States (Srey, et al., 2013).

In produce, microorganisms attach to the surfaces and biofilms develop internally. During the
washing process, biofilms resist sanitization and allow bacteria to spread across the produce,
especially via kitchen utensils.This problem is also found in ready-to-eat foods, because the
foods go through limited cleaning procedures before consumption Due to the perishability of
dairy products and limitations in cleaning procedures, resulting in the buildup of bacteria, dairy
is susceptible to biofilm formation and contamination (Kumar, & Anand, 2018). The bacteria
can spoil the products more readily and contaminated products pose a health risk to consumers.

Methods to Control Biofilms

Biofilm control is a difficult problem to solve. The frequency and severity of infections may
grow, which would increase infection-related mortality and morbidity if biofilms are not
properly combated (Algburi, et al. (2017). As a result of the frequent use of antibiotics to treat
biofilm-associated infections, more virulent, antibiotic-resistant bacteria have started to appear,
necessitating the creation of cutting-edge techniques to eradicate them (Fleming, & Rumbaugh,
2017).
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 Small Molecules
A wealth of knowledge gained in the mechanism of biofilm forming process has led to
the creation of small molecules such as dimethylaminohexadecyl methacrylate
(DMAHDM) composite that can inhibit the formation of biofilms. Dentists have
researched this tactic to prevent the growth of S. mutans and S. sobrinus, which cause
caries (Kuang, et al., 2018).
 QS System Blockers
QS is a very unique microbial communication system based on unique biochemicals
produced by the microbes that control the generation of virulence factors and the
development of biofilms, among other biological processes (Paluch, 2020). Therefore,
inhibition of genes and protein factors involved in imparting virulence can be
successfully inhibited by QS system inhibitors (Singh, et al., 2017).
 Probiotics
Live bacteria known as probiotics provide the host with health benefits when given in
sufficient quantities. Probiotics are typically microorganisms that have inhibitory activity
against specific pathogens, adhesion to epithelial cells, and resistance to specific
concentrations of bile and acid (Balcázar, et al., 2018; Didinen, et al., 2018).

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Conclusion and future perspective

Over time, different strategies have been developed to inhibit the planktonic growth of
microorganisms. But the rise of antibiotic ineffectiveness, multidrug-resistant microorganisms,
and recalcitrant infections directed researchers to understand different aspects of microbial
growth and resistance to environmental cues. Most chronic infections are associated with
microbial biofilms due to their potentiality to resist the known antibiotics and survive even in
harsh environmental conditions.

Although people are trying to combat the problems created by biofilms, they are yet come up
with any novel antibiofilm strategy. Researchers should focus on the strategies which are
efficient, ecofriendly, persistent, and cost-effective as well. In this regard, researchers should try
to develop potent antibiofilm agents from natural products and/or an amalgam of phytochemicals
with other physical, chemical, or biological methods to show synergistic effect and do not
contribute towards the enhancement of microbial resistance.

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