Antimicrobial Resistance Among

Biofilm Forming Bacteria In

Domestic Environments
By
Shaikh Faisal Zaman

Plan of Work

Study and understanding of biofilms.

Study of the history of biofilms.

Research on formation of biofilms.

Study of biofilm life cycle and stages of development.

Study of biofilm habitat.

Research on the infectious diseases in humans caused by biofilms.

Antimicrobial resistance in biofilms and introduction of intrinsic and

extrinsic factors in brief.

What is a biofilm?

A biofilm is any group of microorganisms in which cells stick to each

other on a surface.

Eg: Plaques that form on teeth causing tooth decay is a type of bacterial
biofilm, rocks are coated with biofilm in a stream or river, gunk that
clogs drains, etc.

Biofilm forms when bacteria adhere to surfaces in moist environments

by excreting a slimy, glue-like substance.

Sites for biofilm formation include all kinds of surfaces: natural materials
above and below ground, metals, plastics, medical implant materials
even plant and body tissue. A combination of moisture, nutrients and a
surface leads to biofilm formation.

These adherent cells are frequently embedded within a self-produced

matrix ofextracellular polymeric substance(EPS). The cells produce EPS
and are held together by these strands, allowing them to develop
complex three-dimensional, resilient, attached communities. Biofilms
can be as thin as a few cell layers or many inches thick, depending on
environmental conditions.

Biofilm extracellular polymeric substance, which is also referred to

asslime, is apolymericconglomeration generally composed of
extracellularDNA,proteins, and polysaccharides.

A biofilm community can be formed by a single bacterial species, but in

nature biofilms almost always consist of rich mixtures of many species
of bacteria, as well as fungi, algae, yeasts, protozoa, other
microorganisms, debris and corrosion products.

Over 500 bacterial species have been identified in typical dental plaque
biofilms.

Biofilms are also sites where genetic material is easily exchanged

because of the proximity of the cells, thus maintaining a large gene
pool.

Microbial multicellular communities or biofilms come in a great variety

of sizes and shapes, with some of the most common types containing
mushroom-like, pillar-like, hilly, or flat multicellular structures, which
allow cells to form long-term relationships, interact with each other and
establish metabolic cooperation.

Many of the underlying processes are interdependent and require

cooperation between various bacterial species with different metabolic
capacities. Therefore, in biofilms, the participating microbial members
are situated in close proximity seems to be advantageous, since
metabolites can easily be transferred and metabolized further.

The association of bacteria with a surface and the development of a

biofilm can be viewed as a survival mechanism, with bacteria benefiting
by acquiring nutrients and protection from biocides.

In cases of adverse conditions such as desiccation, osmotic shock, or

exposure to toxic compounds, UV radiation, or predators, the microbial
community as a whole can provide protection.

Staphylococcus aureusbiofilm on an indwellingcatheter

Source:
https://en.wikipedia.org/wiki/Biofilm#/media/File:Staphylococcus_aureus_biofilm_01.jpg

Biofilm in kitchen drain pipe

Source: https://www.biofilm.montana.edu/content/kitchen-drainpipe
http://www.asktheecogeeks.com/clean-stinky-drain/

History of Biofilms

Antonie van Leeuwenhoek (1684) was the first to display the "animalcules"
(bacteria) found in plaque scraped from his teeth plaque, and described in a
report to the Royal Society of London: "The numbers of these animalcules in the
scurf of a mans teeth are so many that I believe they exceed the number of men
in a kingdom.

In1940 H. Heukelekian and A. Heller in an issue of the Journal of Bacteriology

wrote a development takes place either as bacterial slime or colonial growth
attached to surfaces, and Zobell (1943) noted an "effect" in seawater and
described many of the fundamental characteristics of attached microbial
communities.

Biofilm science and biofilm engineering have been active fields of study since
sessile communities were first described and named in 1978. The group of Dr.
Costerton, in 1978, used the name biofilms as a more generic term for
microorganisms adhering to wet surfaces in freshwater ecosystems.

Formation of Biofilms

Formation of a biofilm begins with the attachment of free-floating

microorganisms to a surface. These first colonists adhere to the surface
initially through weak, reversible adhesion viavan der Waals forces.

If the colonists are not immediately separated from the surface, they can
anchor themselves more permanently usingcell adhesionstructures such
aspili.

Hydrophobicityalso plays an important role in determining the ability of

bacteria to form biofilms, as those with increased hydrophobicity have reduced
repulsion between theextracellular matrixand the bacterium.

Some species are not able to attach to a surface on their own but are
sometimes able to anchor themselves to the matrix or directly to earlier
colonists. It is during this colonization that the cells are able to communicate
via quorum sensingusing products such as AHL.

 Some bacteria are unable to form biofilms as successfully due to

their limited motility. Nonmotile bacteria cannot recognize the
surface or aggregate together as easily as motile bacteria.
Once colonization has begun, the biofilm grows through a
combination of cell division and recruitment.
Polysaccharidematrices typically enclose bacterial biofilms. In
addition to the polysaccharides, these matrices may also contain
material from the surrounding environment, including minerals,
soil particles, and blood components, such as erythrocytes and
fibrin.
The development of a biofilm may allow for an aggregate cell
colony (or colonies) to be increasingly antibiotic resistant.
Cell-cell communication orquorum sensing(QS) has been shown
to be involved in the formation of biofilm in several bacterial
species.

Aniridescentbiofilmonthesurfaceofafishtank

Source:
https://en.wikipedia.org/wiki/Biofilm#/media/File:Iridescent_biofilm_on_a_fishtank.JPG

Life Cycle of a Biofilm

Free-floating, or planktonic,
bacteria encounter a submerged
surface and within minutes can
become attached. They begin to
produce slimy extracellular
polymeric substances (EPS) and to
colonize the surface.

EPS production allows the

emerging biofilm community to
develop a complex, threedimensional structure that is
influenced by a variety of
environmental factors. Biofilm
communities can develop within
hours.

Biofilms can propagate through

detachment of small or large
clumps of cells, or by a type of
"seeding dispersal" that releases
individual cells. Either type of
detachment allows bacteria to
attach to a surface or to a biofilm
downstream of the original
community.
Source:
http://www.biofilm.montana.edu/node/2390

Stages of Biofilm Development

There are five stages of biofilm development:
Initial attachment
Irreversible attachment
Maturation I
Maturation II
Dispersion

Five stages of biofilm development: (1) Initial attachment, (2) Irreversible

attachment, (3) Maturation I, (4) Maturation II, and (5) Dispersion.
Source:

The first step is the adhesion of pioneer bacteria, with some of the
planktonic or free-floating bacteria approaching the surface (live or alive)
and becoming attached to the boundary layer, the quiescent zone at the
surface where the flow velocity falls to zero. Some of these cells strike and
are adsorbed to the surface for only a finite time, before being deadsorbed,
in a process called reversible adsorption.

This initial attachment is based on electrostatic attraction and physical

forces, but due to not any chemical attachments. Some of these reversibly
adsorbed cells begin to make preparations for a lengthy stay by forming
structures which may then permanently bind then to the surface within the
next few hours, the pioneer cells proceed to reproduce and the daughter
cells, which form microcolonies on the surface and begin to produce a
polymer matrix around the microcolonies, in an irreversible steps.

In the next stage, focal areas of the biofilm dissolve and the liberated
bacterial cells are then able to spread to other locations where new
biofilms can be formed, and the mature biofilm may contain water-filled
channels and thereby resemble primitive, multicellular organisms and the
attachment is mediated by extracellular polymers that extend outward
from the bacterial cell wall.

This polymeric material, or glycocalyx, consists of charged and neutral

polysaccharides groups that not only facilitate attachment, but also act
as an ion-exchange system for trapping and concentrating trace
nutrients from the overlying water. The glycocalyx also acts as a
protective coating for the attached cells, thereby mitigating the effects
of biocides and other toxic substances.

As well as trapping nutrient molecules, the glycocalyx net also snares

other types of microbial cells through physical restraint and electrostatic
interaction.

In a mature biofilm, more volume is occupied by the loosely organized

glycocalyx matrix (75-95%) than by bacterial cells (5-25%).

The base of the biofilm is a bed of dense, with thickness up 5 to 50 m,

composed of a sticky mix of polysaccharides, other polymeric
substances and water, all produced by the bacteria.

Importance of Biofilm Study

Biofilms in nature are generally beneficial and are frequently established

on hydrous solid and semi-solid surfaces, such as soil, rock material, or
the surfaces of animals and plants.

Microbial communities natively populate human mucous membranes

and epithelial surfaces, for example, the gastrointestinal tract, oral
cavity, and skin.

Despite our bodies being colonized with a mixed microbial community of

characteristic compositions and they are important and beneficial to us
as they can degrade nutrients and thereby make these accessible.

They can synthesize some vitamins which we are unable to synthesize

on our own.

These communities play key roles in the development of our immune

systems and in the anatomy of the mucosal surfaces, and also provide
protective functions against exogenous pathogens.

The relationship between the host and its microbial communities is

delicately balanced, but under certain conditions it can break down and
result in infectious diseases.

According to a recent public announcement from the National Institutes

of Health, more than 60% of all microbial infections are caused by
biofilms.

Although the planktonic form of the bacteria has been very useful in
understanding acute infections, chronic ones are more related to the
presence of biofilms, with current research indicating an important role
for bacterial biofilms in recurrent or chronic infection, including those
which are not responsive to a culture-appropriate antibiotic therapy.

Biofilm-growing bacteria cause chronic infections, including foreign-body

infections, that are characterized by persistent inflammation and tissue
damage despite antibiotic therapy and the innate and adaptive immune
and inflammatory responses of the host and persisting pathology.

Some general features of biofilm infections in humans compared with acute

planktonic infections are:

Aggregates of bacteria embedded in a self-produced polymer matrix

Tolerant to both innate and adaptive immune responses

Tolerant to clinically dosing of antibiotics despite susceptibility of planktonic cells

Chronic infections

The surface-associated microorganisms are responsible for a several chronic

infections as: periodontitis, heart valves (endocarditis), in lung infection in
patients with cystic fibrosis (CF) causing chronic bronchopneumonia by
Pseudomonas aeruginosa, child middle-ear infections (caused by
Haemophilus influenzae, for example), in chronic rhinosinusitis, in chronic
osteomyelitis and infections caused by a variety of surgical implants, wound
infection in burn patients, urinary tract infections (caused by Escherichia
coli and other pathogens), in intravenous catheters and stents (caused by
Staphylococcus aureus and other gram-positive pathogens), among others.

The link between the concept of biofilms and chronic infectious disease is
still the subject of a lot of many studies.

Habitat

Biofilms are ubiquitous. Biofilms will form on virtually every nonshedding surface in a non-sterile aqueous (or very humid) environment.

Biofilms can be found on rocks and pebbles at the bottom of most

streams orriversand often form on the surface ofstagnantpools of
water. In fact, biofilms are important components offood chainsin rivers
and streams and are grazed by the aquaticinvertebratesupon which
many fish feed.

Biofilms can grow in the most extreme environments: from, for example,
the extremely hot, briny waters ofhot springsranging from very acidic
to very alkaline, to frozenglaciers.

In the human environment, biofilms can grow inshowersvery easily

since they provide a moist and warm environment for the biofilm to
thrive. Biofilms can form inside water and sewagepipesand cause
clogging and corrosion. Biofilms on floors and counters can make
sanitation difficult in food preparation areas.

Biofilms are present on theteethof most animals asdental plaque,

where they may causetooth decayandgum disease.

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 Rhizobium
on roots, exist symbiotically with the plant.Examples of crop diseases
related to biofilms include Citrus Canker, Pierce's Diseaseof grapes, and
Bacterial Spot of plants such as peppers and tomatoes.

Recent studies in 2003 discovered that the immune system supports

bio-film development in the large intestine. This was supported mainly
with the fact that the two most abundantly produced molecules by the
immune system also support bio-film production and are associated with
the bio-films developed in the gut.

Biofilm inYellowstone National Park

Source:
https://en.wikipedia.org/wiki/Biofilm#/media/File:Iridescent_biofilm_on_a_fishtank.JPG

Thermophilic bacteria in the outflow ofMickey Hot

Springs,Oregon, approximately 20 mm thick
Source:
https://en.wikipedia.org/wiki/Biofilm#/media/File:Thermophilic_bacteria.jpg

Biofilms and Infectious Diseases

Dental plaque

Dental plaqueis an oral biofilm that adheres to theteethand consists of

many species of both fungal and bacterial cells (such asStreptococcus
mutansandCandida albicans), salivarypolymersand microbial extracellular
products. The accumulation of microorganisms subjects the teeth and gingival
tissues to high concentrations of bacterialmetaboliteswhich results in dental
disease.

The biofilm on the surface of teeth is frequently subject to oxidative

stressand acid stress. Dietary carbohydrates can cause a dramatic decrease
in pH in oral biofilms to values of 4 and below (acid stress).A pH of 4 at body
temperature of 37C causes depurination of DNA, leaving apurinic (AP) sites
in DNA,especially loss of guanine.

Dental Plaque

Source: http://www.redrockdental.org/lp/plaque.html

Streptococcus pneumoniae

S. pneumoniaeis the main cause of community-acquired pneumonia and

meningitis in children and the elderly, and of septicemia in HIV-infected
persons. WhenS. pneumoniagrows in biofilms, genes are specifically
expressed that respond to oxidative stress and induce competence.Formation
of a biofilm depends on competence stimulating peptide (CSP). CSP also
functions as a quorum-sensing peptide. It not only induces biofilm formation,
but also increases virulence in pneumonia and meningitis.

Legionellosis

Legionellabacteria are known to grow under certain conditions in biofilms, in

which they are protected againstdisinfectants. Workers incooling towers,
persons working inair conditioned roomsand people taking ashowerare
exposed toLegionellaby inhalation when the systems are not well designed,
constructed, or maintained.

Source: http://biomedfrontiers.org/infection-2013-dec-2/

Source: http://m2002.tripod.com/legionellosis.htm

Antimicrobial resistance in biofilms

Despite decades of research, very little is known about the molecular

mechanisms of antibiotic resistance in biofilms. Although several
theories have been proposed, the precise mechanism of how this
sensitivity is altered has still not been clarified.

Nevertheless, it is possible to separate these mechanisms into intrinsic

(or innate) and extrinsic (or induced) resistance factors to biofilms.

Also, because of the heterogeneous nature of biofilms, it is likely that

multiple mechanisms of antimicrobial resistance occur.

However, additional mechanisms must also exist to be able account for

increased biofilm antibiotic resistance.

In the traditional antibiotic resistance of planktonic bacteria, usually

involves inactivation of the antibiotic, modification of targets, and
exclusion of the antibiotic. These actions typically require the acquisition
of specific genetic factors, such as genes for -lactamase or efflux
pumps.

One of the most important aspects of bacterial biofilm formation is the

increased resistance of the constituent microbes to antibiotics and other
stressors.

The structural nature of the biofilms and the characteristics of the

sessile cells, produce resistance towards the antimicrobial agents,
leading to a protected environment against adverse conditions and the
hosts defenses.

Video on Bacterial Biofilms

Conclusion

During their evolution, bacteria have been able to develop successful

strategies for survival, which include attachment to surfaces and the
development of protective biofilms where bacteria behave very
differently to the free-floating types.

These successful strategies make it difficult to control biofilm growth,

with a biofilm providing bacteria with a 10- to 1,000-fold increase in
antibiotic resistance compared to free ones.

Due to the heterogeneous nature of biofilms, it is likely that multiple

mechanisms of antimicrobial resistance are useful in order to explain
biofilm survival in a number of cases, with antibiotic resistance being
the result of an intricate mixture of intrinsic and extrinsic factors.

Bibliography

http://www.formatex.info/microbiology3/book/736-744.pdf

http://www.biofilm.montana.edu/node/2390

https://en.wikipedia.org/wiki/Biofilm

http://web.stanford.edu/~amatin/MatinLabHomePage/Biofilm.htm

http://www.sciencedirect.com/science/article/pii/S0924857910000099

http://www.ncbi.nlm.nih.gov/pubmed/20149602

http://www.medscape.com/viewarticle/807731_4

Future Plan of Work

Further research on antimicrobial resistance of bacterial biofilms.

Research on intrinsic and extrinsic factors of resistance to antibiotics in

biofilms.

Study of biofilm formation in domestic environments.

Advantages and disadvantages of biofilm in domestic environments.

Preventive measures to be taken to prevent growth of biofilms on

substances.

THANK YOU