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.

Study of biofilm life cycle and stages of development.

Importance of biofilm study.

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.

Measures to be taken to prevent biofilms in domestic environments.

Conclusion.

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.

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.

Biofilms come in a great variety of shapes and sizes:- mushroom-like,

pillar-like, hilly, or flat multicellular structures.

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.

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) - 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.

Zobell (1943) noted an "effect" in seawater and described many of the

fundamental characteristics of attached microbial communities.

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.

Aniridescentbiofilmonthesurfaceofafishtank

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

Life Cycle of a Biofilm

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:

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.

They can degrade nutrients and thereby make these accessible.

They can synthesize some vitamins which we are unable to synthesize

on our own.

They 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.

Habitat

Biofilms are ubiquitous. Biofilms will form on virtually every non-shedding

surface in a non-sterile aqueous (or very humid) environment.

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 domestic environment, biofilms can grow inshowers. Biofilms can form
inside water and sewagepipesand cause clogging and corrosion.

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.

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 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

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.

ANTIMICROBIAL
RESISTANCE
INTRINSIC
The biofilm matrix
may act as a
diffusion barrier

Establishment of
microenvironment
s within biofilms

Differentiation into
persister cells

Increased
production of
oxidative stress

EXTRINSIC

Intrinsic or innate resistance factors to biofilms

The intrinsic factors of resistance are activated as part of the biofilm

developmental pathway, which are integral parts of the biofilm structure and
physiology resulting from conversion to a biofilm lifestyle.

The influences of several different intrinsic biofilm factors affecting antibiotic

resistance have been identified. For example: the biofilm matrix may act as a
diffusion barrier; microenvironments within biofilms can be established; small
subpopulations of bacteria within the biofilms may differentiate into
persisters; an increased production of oxidative stress causes might changes
in the physiology of bacteria; and an antagonist of antibiotics and degradation
mechanisms may be active in some parts of the biofilms.
The biofilm matrix may act as a diffusion barrier

Biofilms can act as physical diffusion barriers to prevent antibiotics from reaching their
targets. Antibiotic was shown to be able to penetrate these structures through a thick
mixture of exopolysaccharide, DNA, and protein in order to reach the targets.

However, this mixture was not able to achieve an effective concentration in some all parts
due to the physical and/or chemical properties of the matrix, which resulted in an apparent
increase in resistance.

Recent mathematical modeling has predicted that while limited antibiotic diffusion may lead
to the death of the outer layer of bacteria, it also stimulates subpopulations of bacteria
buried deeper within the biofilm to enact adaptive changes, thereby countering the insult.

However, limited antibiotic diffusion does not appear to be a universal trait shared by all
biofilms, and, there is conflicting data about whether the biofilm matrix is a major
contributing factor influencing biofilm resistance.

Establishment of microenvironments within biofilms

The depletion of nutrients and oxygen inside biofilms might cause an altered metabolic
activity and lead to slow growth of the bacteria. Several studies have revealed oxygen
limitation and the presence of hypoxic zones deep within biofilms, with nutrient diffusion
through biofilms being restricted.

Inspection of environmental as well as in vitro biofilms has revealed that the oxygen
concentration may be high at the surface, but low in the centre of the biofilm where
anaerobic conditions may be present.

Likewise, growth, protein synthesis and metabolic activity are stratified in biofilms, for
example, there is a high level of activity at the surface but a low level in the centre, resulting
in slow or no growth. This fact is one of the explanations put forward for the reduced
susceptibility of biofilms to antibiotics.

In general, all antimicrobials are more effective in killing rapidly growing cells. For example,
penicillin and ampicillin have an absolute requirement for cell growth to occur in order to kill,
be able to, with the mortality rate being proportional to the rate of growth.

Therefore, slow growth undoubtedly contributes to biofilm resistance to the killing of

bacteria. In addition, slow growth is a major factor in the increased resistance of stationary
planktonic cells to being killed.

 Differentiation into persister cells

Persister cells are considered to be either nongrowing or slow-growing, and also have a
greatly reduced susceptibility to antibiotics.

In the persisters theory, these small subpopulations of bacteria within the biofilms
differentiate into dormant cells, are able to survive extreme antibiotic treatment and have
been hypothesized to be the result of phenotypic variations rather than due to stable genetic
changes.

The formation of persisters might represent a common mechanism used by a wide range of
bacteria during biofilm formation. This persistent population within biofilms may drastically
inhibit the complete eradication of the biofilms, even after prolonged, high-level antibiotic
treatment.

However, it is unclear what relationship exists between planktonic persisters and biofilm
resistance, and the mechanisms(s) by which persisters form and/or confer increased
antibiotic tolerance are still unknown.

Increased production of oxidative stress

Oxidative stress is caused by an imbalance between the production of oxidants, such as the
free radicals, peroxide and nitric oxide, with the levels of antioxidant defenses.

A disturbance in the prooxidant-antioxidant balance in favor of the overproduction of

reactive oxygen species (ROS) can result in damage to the cellular components, including
the matrix, DNA, proteins and lipids.

Oxidative stress is considered to cause enhanced mutability in biofilms. Recent data suggest
that microcolonies structures, due to endogenous oxidative stress, are specific sites within
biofilms where enhanced genetic adaptation and evolutionary change take place.

In addition, Boles and Singh showed that endogenous oxidative stress in biofilms promotes
antibiotic resistance and that the addition of antioxidants reduces the diversity in biofilms.

Extrinsic or induced resistance factors to biofilms

Other groups resulting from transcriptional induction by treatment with

antibiotics agent itself, is extrinsic or induced resistance factors.

The mutation frequency of biofilm-growing bacteria is significantly increased

compared with planktonically growing isogenic bacteria and there is an
increased horizontal gene transmission in biofilms.

Bacterial cells in biofilms may simultaneously produce enzymes that degrade

antibiotics, have antibiotic targets of low affinity and over express efflux pumps
that have a broad range of substrates.

The source of -lactamase in biofilms has been considered to come from the
layer of lysed bacteria resulting from exposure to antibiotic, and is related to
the release of defensive enzymes into the extracellular space.

Bacteria expressing a high level of chromosomal -lactamase in biofilms could

be exposed to a reduced concentration of -lactam antibiotics, owing to the
accumulation of the enzyme in the polysaccharide matrix.

The extracellular -lactamase may inactivate the antibiotic as it penetrates,

thereby protecting the deeper-lying cells.

These induced factors may work synergistically with intrinsic factors to

enhance survival in the face of strong antimicrobial stresses.

Four hypothesized biofilm resistance mechanisms. 1 The antibiotic (squares)

penetrates slowly or incompletely; 2 a concentration gradient of a metabolic
substrate or product leads to zones of slow or non-growing bacteria (shaded
cells);
3 an adaptive stress response is expressed by some of the cells (marked cells);
4 a small fraction of the cells differentiate into a highly protected persister
Source: http://www.urbanfischer.de/journals/ijmm
state (dark cells).

ANTIBIOTIC

MICROORGANISM

PENETRATIO REFERENCE
N?

Piperacillin

P.aeruginosa

No

Hoyle et al., 1992

Rifampin

S.epidermis

Yes

Dunne et al.,
1993

Vancomycin

S.epidermis

Yes

Gentamicin

P.aeruginosa

No

Yasuda et al.,
1993

Latamoxef

E.coli

Yes

Jouenne et al.,
1994

Ciprofloxacin

P.aeruginosa

Yes

Suci et al., 1994;

Vrany et al., 1997

Amikacin

P.aeruginosa

No

Shigeta et al.,
1997

Experimental measurements of antibiotic penetration into biofilms. The criterion for

penetration was attainment of 30% of the applied antibiotic concentration (or 30% of the
antibiotic concentration determined in a sterile control) during the test duration.
Source: http://www.urbanfischer.de/journals/ijmm

Measures For Prevention of Biofilm

Formation In Domestic Environments

Prevention of biofilm formation may be accomplished by avoiding conditions that lead to cell
attachment and selecting conditions that make the environment unfavorable for microbial
growth.

Regularly-scheduled cleaning and keeping surfaces free of moisture and residue (bacteria
food) is the best defense against biofilms. Once established, biofilms are difficult to get rid of.
Even if the surface looks clean, biofilms can cling tenaciously to out of the way areas.

Studies show that due to their protective matrix, spraying with bleach or another type of
antimicrobial, or a shower or toilet bowl cleaner, and then just rinsing, isnt going to remove
them. Antimicrobial chemicals cannot penetrate the biofilm to kill all bacteria.

Like dental plaque, biofilm must be agitated, broken up, and removed before an antimicrobial
chemical (in the example of plaque - mouthwash) will be effective.

Biofilm removal and inactivation is achieved by combining proper cleaning and sanitizing
agents, adequate exposure time, proper temperature and mechanical action.

This combination dissolves the biofilm and the organic material to which it adheres, allowing
the sanitizer to inactivate the released, sensitive cells. Extensive scrubbing with proper
chemicals is important in biofilm removal.

 Some basic recommendations for removing biofilm from a shower

or tub:
Regular cleaning is a must; weekly is preferred.
Remove soap scum and soil by administering a shower/bathroom
cleaner according to directions. Scrub to remove biofilm and expose
clean surfaces.
After removing the biofilm, it is important next to administer a
disinfectant and let it sit for a few minutes (follow label directions) to
kill any remaining bacteria, before rinsing again.
Due to the porous nature of tile grout, bacteria can remain untouched
in nooks and crannies causing a quick return of the biofilm.
Administering steam vapor will assist in reaching and killing those hard
to reach bacteria, allowing a longer time between biofilm appearances.
Targeted use of an oxidizer like household bleach or oxygen bleach,
used according to directions, may remove discolorations caused by
biofilms.
Towel dry shower surfaces after each cleaning to aid in inhibiting
biofilm growth.

Coating agent

Coating method

Mechanism

Antibiotics

Non-covalent, covalent bonding

Bactericidal/Bacteriostatic

Silver

Plasma deposition, sol-gel coating, wetchemical coating

Bactericidal

Furanones

Physical adsorption, covalent bonding

Bactericidal/Bacteriostatic

QAS

Covalent bonding

Inhibition of bacterial adhesion and viability

Silica nanoparticles with QAS

Covalent bonding

Bactericidal/Bacteriostatic

TMS

Plasma coating deposition with covalent

bonding

Anti-adhesion

PLL-g-PEG

Physical adsorption & covalent coupling

Anti-adhesion

pCBMA

Zwitterionic surfaces grafted via radical

polymerization

Anti-adhesion

Silica colloids/Silane xerogel

Synthesis of superhydrophobic coating

Anti-adhesion

Submicron surface textures

Physical surface roughness modification

Anti-adhesion

Selenocyanatodiacetic acid

Covalent bonding

Anti-adhesion

Polymer brush coatings

Surface grafting

Anti-adhesion

Surface modification approaches that can inhibit biofilm formation

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794791/

Video on Bacterial Biofilms

What Are Bacterial Biofilms A Six Minute Montage.mp4

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.

Some mechanisms of resistance appear to be intrinsic to growth in a biofilm,

with the inhibited diffusion through the matrix, reduced metabolism by
nutrient limitation, formation of dormant persisters, and increased
production of oxidative stress all appearing to impact on the development of
a protective environment within the biofilm; in addition to extrinsic factors
such as -induced gene expression by antibiotics in biofilm cells.

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.

It is clear that much more research is needed to reveal additional and/or

novel antibiotic-induced factors in biofilms, as the multifactorial nature
of biofilm antibiotic resistance has hindered identification of these
pathways, with much still being unknown about the induced factors in
biofilm resistance.

Discovery of these factors should lead to new and better treatments for
biofilm related infections. New strategies are required to overcome
extreme biofilm antibiotic resistance through the development of novel
therapies aimed at disrupting biofilms and killing the constituent
bacteria, with manipulation of intrinsic and extrinsic resistance
pathways providing much promise for future treatment of biofilm
infections.

Proper cleaning and sanitation work best for biofilm prevention while its
removal is only necessary if prevention fails.

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