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Revista Romn de Medicin de Laborator Vol. 25, Nr.

1, Ianuarie, 2017 9

Review

DOI: 10.1515/rrlm-2017-0001

Microbial biofilm in human health


- an updated theoretical and practical insight
Biofilmul microbian n medicin - o perspectiv teoretic i practic

Monica Licker1,2, Roxana Moldovan1, Elena Hogea1,3, Delia Muntean4,*,


Florin Horhat1,2, Luminia Baditoiu1,5, Alexandru Florin Rogobete1,
Emil Trziu6, Csilla Zambori1
Victor Babe University of Medicine and Pharmacy Timioara, Romania 2Pius Branzeu
1

Emergency, Clinical, County Hospital Timioara, Romania, 3Victor Babe Clinical Infectious
Diseases Hospital Timioara, Romania, 4Department of Microbiology, Victor Babe University of
Medicine and Pharmacy Timioara, Romania, 5Regional Centre of Public Health Timioara, Romania,
6
Banats University of Agricultural Sciences and Veterinary Medicine, King Michael I of Romania,
Timioara, Romania

Abstract
The term biofilm designates an aggregate of microorganisms belonging to one or more species which adhere to
various surfaces but also to each another. These microbial communities are included and interconnected within an
organic structure known as slime, composed of protein substances, polysaccharides, and DNA.
The Center for Disease prevention and control considers infections with bacteria in biofilms among the 7 most im-
portant challenges which must be overcome in order to improve the safety of health services. The risk of microbial
biofilm development exists for a long list of medical devices and equipment, as well as in certain diseases such as
cystic fibrosis. An aggravating aspect is represented by the almost 1,000 times higher antimicrobial resistance of
bacteria growing and multiplying within biofilms. Thus, in case of biofilm-infected medical devices, the resistance
to antimicrobial treatments requires the removal of the device which essentially means the failure of the exploratory
or therapeutic intervention in question.
The role of microbial biofilms in medical pathology is a subject that raises interest for both researchers and cli-
nicians in order to establish new methods for prevention and treatment of biofilms. This paper is intended as
an overview in the management of microbial biofilms, presenting future insights, with technological progress in
microscopy, molecular genetics, and genome analysis. Therefore the present paper will focus on describing the
mechanisms involved in biofilm development, biofilm related infections, methods of detection and quantification of
microbial communities and therapeutical approaches.
Keywords: biofilm; medical devices; microscopy.

* Corresponding author: Delia Muntean, Department of Microbiology, Victor Babe University of Medicine
and Pharmacy, Str. E. Murgu Nr. 2, Timioara, Romania, e-mail: deliacristimuntean@yahoo.com
10 Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017

Rezumat
Termenul de biofilm desemneaz agregate de una sau mai multe specii microbiene aderente la diferite suprafee, n
care celulele sunt, de asemenea, unite i ntre ele. Aceste comuniti microbiene sunt incluse i solidarizate ntr-o
structur organic desemnat drept slime n a crei componen intr substane proteice, polizaharide, ADN.
Centrul pentru Controlul i Prevenia Bolilor consider c infeciile cu bacterii din biofilme se situeaz printre cele
mai importante 7 provocri ce trebuie depite, pentru creterea siguranei serviciilor de sntate.
Riscul dezvoltrii de biofilme bacteriene exist pentru o lung list de dispozitive i echipamente medicale, precum
i afeciuni, cum ar fi fibroza chistic. Un aspect agravant l reprezint rezistena la antibiotice, care este de pn la
de 1000 de ori mai mare n cazul bacteriilor nglobate n biofilme. Astfel, n cazul dispozitivelor medicale infectate,
rezistena la tratamentul antibacterian duce la necesitatea ndeprtrii acestora, ceea ce reprezint de fapt eecul
respectivei intervenii medicale exploratorii sau terapeutice.
Rolul biofilmelor microbiene n patologia medical reprezint un subiect care polarizeaz n egal msur inte-
resul clinicienilor i al cercettorilor, n vederea identificrii de noi metode de prevenie i tratament al acestora.
Acest articol dorete s fac un rezumat al managementului biofilmelor microbiene, cu prezentarea perspectivelor,
a progreselor nregistrate n microscopie, genetica molecular i analiza genomic. Prin urmare, lucrarea de fa
se va concentra pe descrierea mecanismelor implicate n formarea biofilmului, infeciilor produse de biofilme, me-
todelor de detectare i cuantificare a comunitilor microbiene i a noilor abordri terapeutice.
Cuvinte cheie: biofilm; dispozitive medicale; microscopie.
Received: 8th August 2016; Accepted: 12th December 2016; Published: 31th December 2016.

Introduction Acinetobacter baumannii, Pseudomonas aerugi-


nosa and Enterobacter spp. Additionally, biofilm
Van Leeuwenhoek was the first to discov-
formation apparently plays a role in dental caries,
er minute living creatures, using lenses made
with the proven involvement of bacteria such as
by himself after he examined saliva, leaves of
Streptococcus sobrinus and Streptococcus mutans.
plants, faeces, soil samples, etc. He also collect-
Another interesting area requiring further research
ed portions of his dental plaque and used the
is represented by the mechanisms of biofilm de-
above-mentioned lenses to examine the material
velopment in cases of implant contamination by
in which he observed minute organisms which
microorganisms such as Proteus mirabilis (3).
he called animalcules (1).
The Center for Diseases prevention and
Much later, in 1976, Marshall detected very
control (CDC) listed biofilm-related pathology
fine extracellular polymer fibrils which he iden-
among the most important safety challenges
tified as means of bacterial adherence to various
confronting health care systems and consequently,
surfaces. In 1978, the term biofilm was formal-
research work focusing on understanding the
ly introduced by Costerton (2), who observed
intimate mechanisms of biofilm formation and
that communities of bacteria found in aquatic
functioning. According to CDC experts, such
systems were enclosed in a glycocalyx matrix,
research involving innovative techniques in
which was shown to mediate adhesion.
microscopy and molecular biology helped the
In the last few years, there has been an in-
scientific community to better understand the
creased focus on the biofilm generating potential
structure and functioning of biofilms, with
of a group of clinically relevant bacteria, caus-
important advances in the management of
ing high mortality, abbreviated with the acronym
infections caused by biofilms. The percentage of
ESKAPE which stands for Enterococcus faecalis,
biofilm-related health care-associated infections
Staphylococcus aureus, Klebsiella pneumoniae,
has been estimated at over 65% (4).
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According to the European Society of Clinical initial stage. In other words, should any factors
Microbiology and Infectious Diseases Biofilms act to detach the microbes from the respective
study group (ESGB), new methods are necessary surface during this early stage, biofilm forma-
for studying biofilm-associated infections in hu- tion would be prevented. Whenever there is no
mans. Such methods are only used in specialized external early action taken against microbial at-
research laboratories. The ultimate goal of ESGB tachment to that surface, the adherence will in-
is to improve the results of prophylaxis and thera- crease and tend to become permanent due to the
py of biofilm-associated infections in humans. In involvement of cell adhesion structures e.g. pili
order to obtain these goals, ESGB realizes that a (1, 2, 3, 7).
multidisciplinary approach is necessary, including Thus, in the case of an implanted medical de-
scientists from basic and environmental microbi- vice or equipment which has been contaminated
ology, as well as molecular microbiology (5, 6). by microorganisms, the chance of further biofilm
development with consecutive infection depends
Biofilm definition on a number of variables. As stated above, the
presence of microorganisms on the device must
The present definition of a biofilm could be for-
last long enough in order for the initial stage of
mulated as an aggregate of one or more microbial
weak, reversible attachment to be transformed
species which adhere to various surfaces but also
into the quasi-permanent adherence. The num-
to one another. These microbial communities are
ber and types of cells in the exposure environ-
included and interconnected within an organic
ment of the implanted device, the liquid flow
structure designated with the term of extracel-
rate (in the case of lumen devices), and the phys-
lular polymeric substance (EPS), also known as
ico-chemical structure of the implanted material
slime, composed of proteic substances, poly-
may be listed among the most important varia-
saccharides, and DNA (1,2,3).
bles involved. To sum up, the main stages of bi-
There are several common characteristics of
ofilm initiation and development may be defined
biofilms developing on diverse solid areas e.g.
as follows: microbial adherence, colony for-
heterogeneous structure, multiple interrelations
mation, biofilm maturation and final dispersion
between the components, diverse genetic fea-
(Figure1). During each of these stages, micro-
tures, strong, irreversible attachment to living or
organisms may benefit from internal or external
inanimate surfaces (1). The rising occurrence of
supportive structures (pili, DNA or exopolysac-
biofilm-related pathology is explainable by the
charides) (1, 2, 3, 7, 8).
increasing frequency of exploratory and/or ther-
apeutic procedures involving the use of implant-
Bacterial growth and multiplication sup-
ed medical devices.
ported by biofilm
Mechanisms of Biofilm generation and Biofilm formation is a naturally occurring phe-
development nomenon encountered in the external environ-
ment and impacting various aspects of human
The first stage of biofilm generation requires mi-
life, such as contamination of foods, corrosion
croorganisms to adhere to a surface. The adhe-
and/or obstruction of pipes, etc. Similarly, such
sion is initially weak, as it is achieved through
phenomena also occur within the human body
van der Waals forces; an important aspect is the
resulting, for instance, in dental plaque forma-
reversible character of biofilm generation at this
tion, mastitis, otitis, pneumonia, urinary tract in-
12 Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017

Free floating
planktonic phase

Biofilm

Attachment
Dispertion
Microcolonies
Extracellular Maturation
matrix

Figure 1. Stages in biopfilm development

fections (UTI), osteomyelitis, as well as biofilm infections and biofilm related waterborne diseases.
contamination of implanted medical devices and In cystic fibrosis patients the airways are
equipments. The most frequently involved mi- filled with an adherent and consistent mucus
croorganisms responsible for biofilm formation favored by biofilms, resulting in severe breath-
on implanted devices include Gram-positive ing problems. Pseudomonas infections often
bacteria (GPB), Gram-negative bacteria (GNB) result in antibiotic-resistant biofilms. Moreover,
and fungi (Table 1). Whenever such microor- in such cases biofilms might contain virulence
ganisms grow and multiply within biofilms, sev- factors, such as the major polysaccharide of
eral characters might differ as compared to their P.aeruginosa matrix (alginate), with consecutive
counterparts not being hosted by biofilms. This pulmonary lesions (3, 9).
is mainly due to the protection provided by bio-
film components which allow bacteria to interact Wounds
with mutual advantages e.g. higher resistance to
Biofilms are commonly found in chronic wounds,
detergents and antimicrobials granted by the ex-
being present either at the surface or within the
tracellular matrix acting as a protecting shield (1,
profound layers (as is the case of P.aeruginosa
2, 3, 7, 5, 9, 10).
biofilms) and contributing to delayed wound
Therefore, biofilms are found to be involved
healing. The microbiological diagnosis of these
in a wide variety of microbial infections which can
infections is usually difficult to achieve by
be classified into three main categories: biofilm
simply swabbing the wound and culturing the
organ-related infections, biofilm implant-related
sample (10,11).
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Table 1. Predominant microorganisms responsible for biofilm formation (1-10, 23, 26, 48)
No Microorganisms Location and/or effect of biofilm associated infections
1. GPB Staphylococcus aureus Cardiac valves (endocarditis)
Catheters
Protheses
Eye infections
Device associated infections
Coagulase negative Cardiac valves (endocarditis)
staphylococci Catheters
UTI
Device associated infections
Streptococcus mutans Dental caries
Periodontitis
Gingivitis
Streptococcus viridans Catheters
Device associated infections
Streptococcus sobrinus Dental caries
Periodontitis
Gingivitis
Enterococcus faecalis Cardiac valves (endocarditis)
Catheters
UTI
Device associated infections
diphteroids Cardiac valves (endocarditis)
2. GNB Escherichia coli UTI
Klebsiella pneumoniae Cardiac valves (endocarditis)
Proteus mirabilis Device associated infections
Enterobacter spp.
Serratia marcescens Eye infections
UTI
Pseudomonas aeruginosa Cystic fibrosis
Wounds
Eye infections
UTI
Cardiac valves (endocarditis)
Waterborn infections
Device associated infections
Acinetobacter spp Device associated infections
3. Other Legionella pneumophila Waterborn pulmonary infections after inhalation of con-
bacteria taminated aerosols
4. Fungi Candida albicans Device associated infections
Cardiac valves (endocarditis)
5. Amoeba Eye infections
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There is a global increase of chronic wounds Biofilms - a great threat to implants


especialy in obesity and diabetes patients. These
As previously stated, biofilm formation signif-
patients are particularly prone to be colonized by
icantly influences the evolution of implanted
a high number of bacterial species (12).
medical devices: intravenous catheters, prosthet-
Dower et al. described biofilms already pres-
ic joints, breast and other implants, contact lens-
ent on wound drains removed even after only 2
es, artificial cardiac valves, or pacemakers. This
hours after placement and they observed that
also applies to dental implants which need to be
even when increased attention to sterility was
protected as well as possible from contamination
involved upon placement of wound drains, the
with the extremely rich oral bacterial flora, in or-
amount of biofilm was not significantly lower.
der to avoid infection with consecutive implant
Their conclusion was that within 2 hours after
failure. Once microbial colonization occurs on
drain placement on closed suction wounds, bio-
such medical devices, chronic, slowly evolving,
films will develop (13).
persistent infections may develop. The incidence
Dental plaque of such complications is expected to rise with the
increased use of implanted medical devices and
Biofilms play an important role in dentistry equipments (3, 10, 16, 17, 18).
with more than 700 bacterial species in the den-
tal plaque where mixed biofilms are present. Biofilm-related endocarditis
The unique conditions in the oral cavity includ- In patients with artificial cardiac valves, micro-
ing moisture, thermic variations, presence of bial colonization of these prosthetic devices and
hard tissues, as well as the presence of carbon of the surrounding tissues with further biofilm
and nitrogen, make this a suitable environment development results in prosthetic cardiac valve
for many microbial species. The bacterial com- endocarditis, which is a severe complication.
ponence of biofilms is dependent on factors These infectious conditions may originate
within this complex and variable environment. from the bacterial flora on the patients skin but
For example, the combined action of intraoral also from other implanted devices e.g. central
acidity (e.g. acid foods on dental surfaces) and intravenous catheters (CVC) or dental implants
the dental adherence and growth of S.sobrinus contaminated with S.epidermidis, S.aureus,
and S.mutans, which are acid-tolerant bacteria, Streptococcus spp., enterococci, diphtheroids,
highly contribute to various oral diseases such GNB, Candida spp. etc. Moreover, biofilm bac-
as dental caries, periodontal disease, gingival teria may sometimes enter the bloodstream with
inflammation, etc. Bacterial inter-communi- consecutive dissemination into other organs and
cation is a paramount process supporting col- tissues (1, 3).
onization and biofilm formation on the dental Biofilm-related ocular infections
enamel. Once biofilm formation has been con-
A number of materials and devices used in oph-
solidated, the protective shield provided by
thalmology have been reported to be exposed
non-bacterial biofilm components will further
to biofilm development. Among the most fre-
support information exchanges between bio-
quently involved devices, materials used for the
film bacteria, contributing to important aspects
surgery of retinal detachment (scleral buckling)
such as resistance to antiseptics and/or antibiot-
were cited together with contact and intraocu-
ics (3, 14, 15).
lar lenses, materials used for sutures, etc. The
Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017 15

infecting moment most frequently identified, Biofilm contamination of indwelling medi-


is during surgical procedures involving the cal devices
above-mentioned devices and/or materials Bacteria commonly isolated from indwelling
which can become contaminated and further medical devices include: E.faecalis, S.aureus,
infected with the development of biofilms (1, S.epidermidis, Streptococcus viridians, E. coli,
10, 19, 20). Also, in the case of eye contact K.pneumoniae, P.mirabilis, and P.aeruginosa or
lenses, the moisture required for their storage yeasts (1, 10).
may represent a risk factor for biofilm develop- Biofilm quantification in the case of CVC
ment with consecutive infections of the ocular is achieved by the so-called roll-plate method
cornea. Literature data show that between 20 which involves the removal of the catheter tip
and 80% of persons wearing contact lenses may which is then rolled onto the surface of a non-se-
acquire corneal infections with fungi, bacteria, lective culture medium, which is thus inoculat-
amoebae, originating from biofilm contamina- ed with the microbial contaminants, if any. The
tion of these devices and the cases in which number of colony forming units (CFU) on the
they are stored (1). In a research conducted by culture medium will represent a quantification of
Szczotka-Flynn et al., lotrafilcon A silicone hy- the microbial load of the catheter tip, this being
drogel contact lenses were found to be contam- considered as a reference technique for this pur-
inated with both reference and clinical strains pose. The downside of this method is that sam-
of Serratia marcescens, P.aeruginosa and pling is limited to the catheter tip and it does not
S.aureus, developing within biofilms and which include the internal surface of the CVC and thus,
resisted to solutions of biguanide compounds, there is no information regarding the microbial
common care solutions for such devices (18). contamination and/or biofilm formation within
Biofilm contamination of urinary catheters the catheter lumen. As a consequence, this meth-
The contamination of latex or silicone tubes od has low levels of sensitivity and predictive
used for urinary catheterization may occur value for detecting bacteremia cases originat-
during the insertion, with biofilm develop- ing from biofilm contaminated CVCs. There is
ment both on the outer and internal surfaces also a quantitative limitation of this technique as
of these devices. The list of microorganisms no more than 1,000 CFU/ tip can be detected.
most frequently involved in biofilm develop- Alternatively, preliminary sample preparation by
ment in the lumen or on the outer surfaces of sonication and vortexing improved quantifica-
urinary catheters includes germs originating tion and the positive predictive value threshold
from the skin (e.g. Staphylococcus epidermid- for septicemia originating from biofilm contam-
is) or from the mucous membranes of the uro- inated CVCs was set at 10 4 CFU / catheter tip
genital area, as well as from the intestinal flora (2).
(E.faecalis, E.coli, P.mirabilis, P.aeruginosa, Endotracheal tubes
K.pneumoniae). A very important factor in- Pneumonia in the ventilated, critically ill pa-
fluencing biofilm formation is the duration of tients remains a complex disease with multifac-
catheterization. A prolonged time of contact torial etiology. Biofilms form on endotracheal
between the biofilm contaminated catheter tubes (ETT) and can impact airway resistance.
will favor a biofilm-related UTI (1, 3, 10, 21, According to Wilson et al., the lifecycle of a
22). biofilm has four stages. Authors concluded that
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advanced biofilms stage (stage IV) is associated implant removal is required (1, 9, 10, 26).
with pneumonia in ventilated patients. The dura- In a study conducted on biofilm contami-
tion of intubation does not appear to be related to nated contact lenses, Brothers et al. found that
biofilm stage (23, 24). the effectiveness of treatments was severely in-
fluenced due to impaired drug diffusion through
Prosthetic joint infection
bandage contact lenses (19).
Usually, GPB, such as staphylococci, are more In a rat model developed by Arad E. et al.
frequently involved. In cases of blood or lymph for silicone breast implants contaminated with
bacterial contamination after surgical interven- methicillin-resistant S. aureus (MRSA), biofilm
tions performed for placement of articular pros- infection proved to be persistent, asymptomatic
theses, microorganisms adhere to the surface of and with high resistance to vancomycin (27).
the implanted medical device in question and In the case of patients with prosthetic cardi-
biofilm generation begins. Such infections often ac valves, antibiotics are prescribed before any
evolve atypically and may escape detection due dentistry procedures to prevent blood stream
to the lack of symptoms, e.g. absence of fever diffusion of oral cavity bacteria and their further
during the early stages and the diagnosis of bio- adherence to the surface of the cardiac valves.
film-related prosthetic joint infection is delayed This antibiotic prophylaxis is extremely impor-
to later stages when local pain appears (10). tant as, once a biofilm contamination and infec-
tion has occurred, antimicrobial treatment might
Biofilms and waterborne infections not suffice and surgery for valve removal is often
Biofilm contamination of healthcare water sys- required.
tems is also possible and requires an active and Microorganisms in biolms are resistant to
continuous risk management. All healthcare fa- antimicrobial agents, but not in the classic sense
cilities should have water safety plans as part (by lactamases production, modication of tar-
of their infection control programme that de- gets, or exclusion of the antibiotic to their tar-
fine responsibilities, design and maintenance, gets). Antibiotic resistance of biofilms is based
monitoring and action plans. Biofilm formation upon the capacity of microorganisms to rely on
and water contamination of the healthcare wa- complement of genes, as a result of microbial
ter systems should be controlled and prevented growth and development within the complex
through some simple interventions (like the use biofilm environment with an additional contri-
of micro-filters, shorter flexible shower hoses, bution of antibiotic pressure. In other words,
systematic walk-through of the water system, biofilm antibiotic resistance can be defined as
etc.) (25). resulting from a combination of genetic and in-
duced factors.
Influence of biofilms on antimicrobial The genetic mechanisms of this biofilm an-
treatment tibiotic resistance are classified into two gener-
al classes: innate resistance factors and induced
The antimicrobial resistance of microorganisms
resistance factors. The innate mechanisms are
living and multiplying within biofilms may be up
considered as activated mechanisms of the bio-
to 1,000 times higher than in the absence of bio-
film development. These factors are considered
films. For this reason, in cases of biofilm contam-
as integral parts of biofilm structure and physiol-
inated implanted devices the antibiotic treatment
ogy and include: decreased diffusion of antibiot-
often fails to solve the infection and surgery for
Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017 17

ics through the biofilm matrix, decreased oxygen synthesis of polysaccharides which favor adhe-
and nutrient availability accompanied by altered sion to solid surfaces as well as colonization.
metabolic activity. These enzyme secretion processes are in fact
Induced resistance factors include those re- triggered by the very contact of bacteria with the
sulting from induction by the antimicrobial agent solid surfaces of implanted devices.
itself. Biofilm antibiotic resistance is mostly The resulting polysaccharide matrix which
manifested as a mixture of innate and induced adheres to the solid surface offers effective pro-
mechanisms (2, 9, 28). tection against phagocytosis. The phagocytes,
One of the most interesting aspects con- while unable to act upon biofilm-protected bac-
cerning the special situation of microorganisms teria will still release high levels of cytokines
growing inside the complex and protective en- and pro-inflammation factors causing inflam-
vironment provided by biofilms is the so-called mation and other cytokine-related lesions in the
quorum sensing. This phenomenon is based adjacent tissues (1, 2).
upon the capacity of bacteria to communicate in
order to quantitatively regulate the biofilm con- Practical approach for biofilm detection
tent. For this, whenever bacterial populations
Standard microbiological sampling is insuffi-
reach a critical threshold, low molecular weight
cient for detecting bacterial biofilm (31). The
molecules are released. This process is involved
traditional approach for biofilms detection in-
in expressing bacterial factors of virulence and it
volves the recovery of live bacteria within the
explains the changed and, sometimes, increased
biofilm, the detection of biofilm by in vitro or
virulence of microorganisms growing inside bi-
in vivo techniques and subsequent identification
ofilms (1). QS operates through a wide range of
and imaging of microbial communities on ana-
signals such as: Oligopeptides (510 amino acid
lyzed surfaces (Table 2).
cyclic thiolactone), N-acyl homoserine lactones
(AHLs), Furanosyl borate (Autinducer-2, AI-2), In vitro techniques for biofilm detection
Hydroxyl-palmitic acid methylester, and Methyl Microtitre plate assays (MTP)
dodecanoic acid. QS inhibition is referred to in- MTP-based assays are the cheapest methods for
hibiting signal synthesis or direct degradation biofilm detection as they involve small amounts
of the signal, inhibition of binding of the signal of reagents while offering the possibility to run
molecule to the receptor and/or inhibition of the a range of tests in parallel. For all these reasons,
signal transduction cascade (29). these tests are best suited for screening (32).
The reasons why these modified phenotypes MTP-based model systems are important in
have not been previously detected might be re- screening the effectiveness of antimicrobials, as
lated to their growth within highly nutritional, well as of disinfectant substances, both synthetic
planktonic environments. and natural (extracted from plants) against bio-
films. Another application of this model would be
Influence of biofilms on the immune system to improve the composition of impregnation ma-
Biofilms provide microorganisms with protec- terials by adapting them to biofilm development
tion against human specific and nonspecific de- by regulation of various parameters (moisture lev-
fence mechanisms. Also, bacteria contribute to el, the temperature of incubation, composition of
the development and growth of their protective growth media, shear stress, the concentration of
biofilm by secreting enzymes which catalyze the oxygen and carbon dioxide) (33).
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Table 2. Practical approach for biofilm detection (2, 8, 11, 15, 16, 19, 25-47)
No Technique Methods
1. Detection of biofilm Microtitre plate assays
by in vitro techniques In vitro flow displacement biofilm model systems
In vitro microfluidic devices
In vitro tissue Culture Plate, Tube method, Congo Red Agar method
2. Detection of biofilm Non vertebrate animal models
by in vivo techniques Vertebrate animal models
In vivo tissue biofilm models
In vivo device related infections biofilm models
3. Quantification of Microtiter plates
microbial biofilm Techniques to determine the total biofilm biomass
The number of viable sessile cells only
The amount of extracellular polymers in the biofilm matrix
Roll-plate technique for central venous catheters
4. Visualization of Scanning electron microscopy
microbial biofilm Epifluorescence microscopy
Confocal laser scanning microscopy
5. Bacterial recovery Sonication and isolation
Polymerase chain reaction
Fluorescent in situ hybridization
Metabolomic profiling and genomic analysis
Multiple diagnostic techniques

In a study conducted by Zambory et al. lated and identified Haemophilus strains were
(34), the authors aimed to assess the in vi- tested for biofilm-forming capacity (35).
tro capacity of planktonic bacterial strains
In vitro flow displacement biofilm model systems
possibly involved in zoonoses to form bio-
films. For this, supragingival samples were Such systems involve an in-flow of nutritive sub-
collected from dogs with dental diseases. stances with the parallel removal of waste. The
Microbiological tests isolated and identified flow displacement biofilm model systems can be
75 strains of Staphylococcus, Streptococcus, subdivided into two groups: either following the
Neisseria and Pasteurella. MTP was further continuous flow stirred tank reactor (CFSTR)
used to assess the capacity of these microbial approach or the plug flow reactor (PFR) ap-
strains to generate biofilms and all the tested proach. An example of flow displacement model
strains tested positive for biofilm formation ca- often used to study the oral biofilms is the con-
pacity, with inter- and intraspecific differences stant depth film fermenter (CDFF). This CDFF
(34). In another study, conducted on adenoid- was used to highlight the effect of some anti-
ectomised children, nasopharyngeal swabs, microbial agents and the influence of surface
and adenoid core were collected and the iso- roughness on biofilm development (36, 37).
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In vitro microfluidic devices have been developed. These models allow re-
Such equipments support biofilm formation un- searchers to study the efficacy of numerous anti-
der different physiological conditions such as microbial agents, the stages of biofilm formation
physiological flow velocities and low fluid-to- and the microbial dissemination to other organs
cell volume ratios. The small sized chambers of and tissues. Among the initial experiments to be
the fluidic devices allow the high-resolution mi- designed, a rat model was developed to study
croscopic biofilm analysis and also offer a high the biofilm generating capacity of S. aureus and
precision environmental control (33). S. epidermidis (41, 42).
Nowadays, various substrates are being test-
In vitro tissue Culture Plate (TCP), Tube method ed for their effects on growth and development of
(TM), Congo Red Agar method (CRA) biofilms using models which involve the subcu-
Afreenish Hassan et al. (38) have studied three taneous implantation of foreign bodies (43, 44).
different methods (TCP, TM, CRA) to detect
In vivo tissue biofilm models
biofilm forming microorganisms isolated from
clinical specimens and to compare these meth- Infections related to biofilm growth and devel-
ods for biofilm detection. They observed that opment have been simulated in living tissues, re-
compared to TM and CRA methods, TCP meth- sulting in the design of biofilm models, with the
od can be used as a general screening model to purpose of overcoming the multiple diagnostic
detect biofilm generating bacteria. and treatment challenges.
In vivo models of tissue induced infections
In vivo techniques for biofilm detection for biofilm study are numerous today such as:
Non vertebrate animal models oral cavity infections (dental caries, periodonti-
In the last years host-microbe interactions and tis), ear, nose and throat infections (otitis media,
immune system responses in non-mammalian rhino sinusitis), lung infections (cystic fibrosis,
models like the fruit fly, Drosophila melano- chronic obstructive pulmonary disease) UTI
gaster were studied, directly related to gut col- (cystitis, pyelonephritis), gastro-intestinal infec-
onization by biofilms. Non-mammalian models tions, wound infections, cardiac infections (en-
are suitable whenever quick, cheap and easily docarditis), osteomyelitis (40).
performable experiments are needed. The lim- In vivo device related infections biofilm models
itations of such models are revealed whenever
Researchers have classified the in vivo models
more complex results are required e.g. immune
of infections originating from implanted med-
reactions in response to biofilm-related infec-
ical devices into two groups. The first group
tions. Also, the brief duration of these models,
of the biofilm models inserts the device (for-
which in some cases is a strong advantage, may
eign-body) in the same organ / position as done
become a drawback which makes them not suit-
in clinical settings e.g. intravascular catheter
able for the research of chronic biofilm-related
models or intrafemoral pins or wires. In the sec-
infections (39).
ond group of biofilm models the foreign body
Vertebrate animal models is inserted in a subcutaneous pocket avoiding
Experiments in quantification of microbial bi- contact with any specific organ; these so-called
ofilm formation on CVC can lead to morbidity subcutaneous models may be defined as tis-
and mortality (40). In order to study biofilm for- sue cage model, e.g. portions of catheters intro-
mation on CVC in vivo, several animal models duced subcutaneously.
20 Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017

Frequently used in vivo devices for biofilm sitivity, specificity, and lower duration, but still,
detection are: vascular catheters, endotracheal cannot discriminate between living and killed
tubes, urinary catheters, orthopedic infections, microorganisms. As a consequence, the risk of
prosthetic joints, vascular grafts, contact lenses, false positive results comes as a disadvantage. In
dental implants (45). order to support a more rapid diagnosis of bio-
film-related infectious complications by PCR-
Quantification and visualization of micro-
based methods, Ryan S. et al. proposed a revised
bial biofilm
algorithm, to guide the plastic surgeon when
As an alternative to techniques involving microti- facing complications that involve biofilms. They
ter plates as growth support, researchers developed have highlighted that the management of recur-
additional methods for quantitative biofilm anal- rent capsular contracture in breast augmentation
ysis such as those determining the total biomass and biofilm reaction to soft-tissue fillers are im-
(i.e. matrix plus living and dead cells, e.g. crystal portant examples of how rapid polymerase chain
violet staining, Syto9 staining), those detecting reaction technology can contribute to build up a
and enumerating only viable sessile cells (e.g. re- clinical algorithm for care that will help to choose
sazurin staining) or the techniques which measure a specific targeted antibiotic treatment (8).
the amount of extracellular polymers in the biofilm More recently, fluorescent in situ hybridiza-
matrix (e.g. dimethyl methylene blue staining) (28). tion, using a fluorescein labeled probe, specific for
Scanning electron microscopy (SEM) is cit- the 16S ribosomal RNA of prokaryotic cells, was
ed among the techniques allowing optimal visu- reported to bind and detect biofilm on surface sam-
alization of the biofilm bacteria within the EPS, ples. Once bound, the biofilm can be detected with
as well as of their direct attachment to the re- CLSM or fluorescent microscopy (17, 31, 46).
spective surface (16, 20, 27, 31). The structure analysis of the biofilm and ex-
For example, for the identification of bio- tracellular matrix exopolysaccharide was done
films in wounds, direct visualization by micros- by CLSM in the tissue of the upper airway and
copy has been proposed, using a modified Congo oral cavity (47, 48). CLSM is superior to SEM
Red solution, 57 SEM, epifluorescencemicros- because it does not require dehydration of the
copy, and confocal laser scanning microscopy sample and consequently, the structure of a bio-
(CLSM) (11). film is better preserved. In a study on tonsillitis
The sampling error might though influence patients, Kania et al. (49) proved CLSM with
the accuracy of results. Preliminary sample double staining to be better than SEM in identi-
processing by sonication improves bacterial re- fying mucosal biofilms.
covery because, following this procedure, the CLSM software (e.g. COMSTAT), developed
EPS is fractured and bacteria are released into by (50) and (51) is used to quantify three-di-
the sample. The next steps follow the classical mensional biofilm images of tremendous val-
bacteriological diagnosis with cultivation on en- ue in quantitative biofilm research. With this
richment media and identification by traditional COMSTAT software, researchers can determine
techniques. While the bacteriological diagnosis the mean thickness, roughness, substratum cover-
remains the golden standard, improvements have age and surface to volume ratio of biofilms (51).
been recently been proposed including the use of Toms I. et al. have analyzed in vivo human
Polymerase Chain Reaction (PCR) for the detec- models of undisturbed oral biofilm by CLSM.
tion of bacterial nucleic acids. Such techniques They have used different types of oral appli-
have advantages related to their increased sen-
Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017 21

ances and substrates which were analyzed by techniques (54, 55). The major challenge is rep-
several microbiological and microscopic meth- resented by the antibiotic resistance of bacteria
ods in combination with CLSM. They have also growing in biofilms (56). There are promising
discussed about a new microscopic technique results of vegetal extracts and essential oils
called confocal endomicroscopy, which can be which apparently could be involved in inhibiting
used for the in vivo microscopic investigation in the synthesis of the peptidoglycan component of
the field of dentistry (52). the bacterial wall (24, 57).
Chronic wounds represent a challenge because For example Dorman and Deans (58) re-
reliable markers are still needed for more accurate ported that phenolic structures of individual oil
tests. At present, some promising methods are be- components presented wide spectrum antibacte-
ing tested such as metabolomic profiling (the study rial effects greatly dependent on their chemical
of small molecular metabolites), and genomic anal- structures. Encouraging results might be expect-
ysis (Quantitative or multiplex PCR) (41). ed from strategies combining antibiotic thera-
According to Deva et al., multiple diag- pies and essential oils. In a research conducted
nostic techniques must be combined in order to by Rodrigues et al. (59), the oil extracted from
improve the diagnosis of biofilm contaminated Croton zehntneri leaves was reported to enhance
medical devices with consecutive biofilm-relat- the effect of gentamicin against P.aeruginosa by
ed infections (31). 42.8% through gaseous contact supporting the
For example for analyzing biofilms by use of essential oils as adjuvants of antibiotic
CLSM, researchers have used ultrasonic units therapies (59).
for a gentle removal of biofilms from joint pros- Regarding the prevention of biofilm for-
theses, internal fixation devices, vascular pros- mation, probiotic substances apparently offer
theses, cardiac pacemakers and defibrillators, further opportunities (60). Encouraging data re-
dental implants, neurosurgical shunts or breast garding probiotics and their antimicrobial effects
implants (27). have been obtained by various research labora-
Sampedro et al. conducted a comparative tories. Probiotics have proved to be effective in
study on spinal implant infection detected by curing diseases such as dental caries, periodontal
conventional tissue culture and by a combined diseases, halitosis and candidiasis (61,62,63).
technique using preliminary sample preparation Zambori et al. (64) have highlighted the
by vortexing and bath sonication in order to dis- antimicrobial potential of probiotics in vitro.
rupt biofilms and release in-growing bacteria. More specifically, it was demonstrated that
The conclusions showed that the preliminary Lactobacillus casei, subsp. casei DG was effec-
implant preparation by sonication followed by tive in killing all dental plaque multidrug-resistant
culture improved the sensitivity of biofilm bac- microbial species, while a mixed Lactobacillus
terial detection and identification (53). acidophilus LA-5 and Bifidobacterium BB
-12 culture had a bacteriostatic effect. It has
Emerging methods for the treatment of also been hypothesized that probiotics and preb-
biofilms iotics combined could act synergistically in sup-
porting oral health (61).
The treatment of biofilm-related infections re-
Cotar et al. demonstrated by real time RT-
mains on the agenda of researchers worldwide,
qPCR that in all P.aeruginosa strains grown in
with certain experimental model treatments
the presence of probiotic culture sterile filtrates,
linked to molecular biology state-of-the-art
22 Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017

the level of QS genes expression was reduced within biofilms. This novel approach of wound
comparatively with those from control cultures. management which includes specially designed
Thereby the soluble molecules secreted by pro- anti-biofilm measures had beneficial effects on
biotics can inhibit the virulence factors regu- wound healing demonstrating the utility of such
lation mechanisms and could represent a new interventions (69).
pathway for pathogenicity and virulence atten- Hazer et al. designed an innovative spine
uation in P.aeruginosa nosocomial strains (65). implant model using modified pedicle Titanium
According to Krespi et al., pulsed laser (Ti) screws with the aim to check for anti-MR-
technology has the ability to generate a pow- SA effects, if any. Implants were inserted in
erful pressure wave sufficient to effectively the lumbar spine of a rabbit, at multiple sites.
disrupt Pseudomonas biofilms in vitro, without The antimicrobial effect was illustrated by the
visible damage to the underlying host structure. inhibition of biofilm formation following the
It also eliminates the ability of the remaining insertion of these modified Ti screws (70).
biofilm to receive nutrients from the external Mechanical removal of the infected area or
environment through its pores and web-like body part, e.g., dental infections is also indi-
channels (66). cated (12).
Mohammad Asnaasharia et al. (67) pro-
posed photodynamic therapy (PDT) as an ef- Conclusions
fective supplement in root canal disinfection.
The cooperation between specialists in studies
For this, the authors involved two PDT-based
focusing on early detection of biofilm-associ-
methods which were checked for their antibac-
ated infections and for the implementation of
terial effects. One of the methods used a light
novel therapies and prevention methods seem
emitting diode lamp (LED lamp, 630 nm) and
attractive perspectives for the management of
the other involved the use of a diode laser (810
microbial biofilms.
nm). Both were tested on E. faecalis biofilms
Resolving pathologies caused by biofilms,
detected on extracted anterior human teeth. The
require additional and/or alternative strategies
PDT using the LED lamp was more effective in
like: the use of probiotics, prebiotics, essential
reducing the number of CFUs of E. faecalis in
oils; developing new methods in order to con-
human teeth.
trol pathogenic bacteria and favour the growth
The attachment and growth of human mi-
of non-pathogenic ones in biofilm communi-
crobial flora biofilm proved to be inhibited by
ties; discovering new animal model systems in
a so-called photo functionalization therapy re-
order to study in vivo polymicrobial biofilms;
sulting in changed surface properties of titani-
characterization of gene expression products of
um implants, as demonstrated by Dorigatti de
biofilm microorganisms, as well as clarifying
Avila et al. (68).
the intimate mechanisms of inter-bacterial ge-
Systemic treatment with linezolid was pro-
netic material exchange within biofilms; math-
posed by Fernndez-Barat et al. to limit biofilm
ematical modeling and computer simulation
development and MRSA burden within ETT, in
development which might contribute to impor-
ventilated pigs with MRSA pneumonia (24).
tant breakthrough results where biofilm-related
Wolcott et al. proposed interventions com-
infections are concerned.
bining debridement with specific anti-biofilm
agents against bacterial phenotypes developing
Revista Romn de Medicin de Laborator Vol. 25, Nr. 1, Ianuarie, 2017 23

Conflicts of interest 3. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu E,


Sintim HO. Biofilm formation mechanisms and targets
for developing antibiofilm agents. Future Med Chem.
The authors declare that they have no conflict of 2015;7(4):493512. DOI: 10.4155/fmc.15.6.
interest.
4. Donlan RM. Biofilms and device-associated infec-
tions. Available from: http://wwwnc.cdc.gov/eid/arti-
Abbreviations cle/7/2/70-0277_article.
EPS ==Extracellular polymeric substance 5. http://www.escmid.org/research_projects/study_
CDC ==Centers for Disease Control and groups/biofilms/presentations_publications/.
Prevention 6. Hoiby N, Bjarnsholt T, Moser C, Bassi GL, Coenye
ESGB ==European Society of Clinical T, Donelli G, et al. ESCMID guideline for the diag-
nosis and treatment of biofilm infections 2014. Clin
Microbiology and Infectious Dis- Microbiol Infect. 2015 May;21 Suppl 1:S1-25. DOI:
eases Biofilms study group 10.1016/j.cmi.2014.10.024.
UTI ==Urinary tract infections 7. Hope CK, Wilson M. Biofilm structure and cell vi-
GPB ==Gram- positive bacteria tality in a laboratory model of subgingival plaque.
GNB ==Gram-negative bacteria J Microbiol Methods. 2006 Sep;66(3):3908. DOI:
10.1016/j.mimet.2006.01.003.
CVC ==Central venous catheters
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MTP ==Microtitre plate assays vival and resistance mechanism of microorganisms.
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CDFF ==Constant depth film fermenter
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TCP ==Tissue culture plate
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PCR ==Polymerase chain reaction infections. APMIS Suppl. 2013 May;(136):1-51.
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Ti ==Titanium film formation on closed suction wound drains. Plast
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