Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Diagnosis of biofilm infections: current methods used,

challenges and perspectives for the future
N.B.S. Silva1 € der3
, L.A. Marques2 and D.D.B. Ro
1 Applied Immunology and Parasitology, Institute of Biomedical Sciences, Federal University of Uberl^ andia, Uberl^
andia, Minas Gerais, Brazil
2 Health Sciences, Medical School, Federal University of Uberl^ andia, Uberl^
andia, Minas Gerais, Brazil
3 Institute of Biomedical Sciences, Federal University of Uberl^
andia, Uberl^
andia, Minas Gerais, Brazil

Keywords
clinical practice, diagnosis of biofilm, in vitro
methods, in vivo methods, MALDI-TOF MS,
omics analysis.
Correspondence
Nagela B.S. Silva, Applied Immunology and
Parasitology, Institute of Biomedical Sciences,
Federal University of Uberl^andia, Av. Ama-
zonas, S/N - Umuarama, Uberl^andia, Minas
Gerais, Brazil.
E-mail: nagela_bernadelli.mg@hotmail.com
2021/1947: received 6 June 2020, revised 1
February 2021 and accepted 23 February
2021
doi:10.1111/jam.15049
Summary
The diagnosis of biofilms continues to be a challenge, and there is no
standardized protocol for such a diagnosis in clinical practice. In addition,
some proposed methodologies are expensive to require significant amounts of
time and a high number of trained staff, making them impracticable for
clinical practice. In recent years, mass spectrophotometry/matrix-assisted laser
desorption ionization time of flight (MALDI-TOF) has been applied it in
biofilm studies. However, due to several problems and limitations of the
technique, MALDI-TOF is far from being the gold standard for identifying
biofilm formation. The omics analysis may prove to be a promising strategy
for the diagnosis of biofilms in clinical laboratories since it allows the
identification of pathogens in less time than needed for conventional
techniques and in a more specific manner. However, omic tools are expensive
and require qualified technical expertise, and an analysis of the data obtained
needs to be careful not to neglect subpopulations in the biofilm. More studies
must therefore be developed for creating a protocol that guarantees rapid
biofilm identification, ensuring greater chances of success in infection control.
This review discusses the current methods of microbial biofilm detection and
future perspectives for its diagnosis in clinical practice.

symptoms that the patient presents are nonspecific (Jamal

Introduction
Biofilms are microbial communities adhered to biotic or
abiotic surfaces and protected by an extracellular poly-
meric matrix; they have been extensively investigated in
recent decades (Costa-Orlandi et al. 2017). It is estimated
that approximately 65% of microbial infections are
caused by biofilm-forming microorganisms, representing
a serious public health problem due to the evasion of the
host immune system and the resistance of most antimi-
crobials used for treatment, increasing the persistence of
the infection (Mooney et al. 2018). Additionally, biofilms
contribute to the increase in morbidity and mortality
rates and hospital costs, making it difficult to treat and
eradicate infections, especially those formed in and on
invasive devices such as catheters and implants (Jamal
et al. 2018).
The diagnosis of biofilm-related infections continues to
be a challenge since in most infections, the signs and
et al. 2018). Although there are several methods for diag-
nosing biofilms in research laboratories, there is no stan-
dardized protocol for such a diagnosis in clinical practice.
Additionally, susceptibility tests to antimicrobials con-
tinue to be performed on planktonic bacteria, not repre-
senting the clinical reality in the case of an infection
associated with biofilms (Ferrer et al. 2016).
This review discusses the current methods of microbial
biofilm detection and future perspectives for its diagnosis
in clinical practice.
Clinical and laboratory diagnosis of microbial
biofilms
In 2014, the European Society for Clinical Microbiology
and Infectious Disease published a guideline to assist
healthcare professionals in the diagnosis of infections
associated with biofilms, with emphasis on clinical and

Journal of Applied Microbiology © 2021 The Society for Applied Microbiology 1

Diagnosis of biofilm infections
laboratory procedures to facilitate diagnosis as well as to
make patient treatment faster and more effective (Høiby
et al. 2015). According to this guideline, the most fre-
quent clinical signs of an infection associated with bio-
films consist of inflammatory reactions, redness, pain,
loss of function and fever. The patient must have a his-
tory of predisposition to infection, such as the use of a
medical device or self-limiting diseases, as well as present-
ing a persistent infection over more than 7 days and fail-
ure of antibiotic therapy. Regarding biofilm laboratory
diagnosis, the guideline suggests some detection methods,
such as electron microscopy and polymerase chain reac-
tion (PCR), where it is possible to identify microbial
aggregates around inflammatory cells and the presence of
mucoid or small cells in positive cultures, indicating
antibiotic recalcitrance (Kamaruzzaman et al. 2018).
In the literature, the methods for diagnosis of infec-
tions caused by free-living bacteria, such as those related
to catheter and chronic wounds, are well established
(Frickmann et al. 2017; Stoica et al. 2017; Rajapaksha
et al. 2019). However, when it comes to the diagnosis of
biofilms, there are few consistent reports and data avail-
able, especially regarding biofilms formed in and on med-
ical devices (Høiby et al. 2015). It is estimated that 2% of
knee and breast implants, 4% of mechanical heart valves,
pacemakers and defibrillators, 10% of ventricular devices
and 40% of ventricular assist devices are affected by bio-
films (Del Pozo 2018). The pathogens are generally
derived from the hands of healthcare workers, contami-
nated water or fluids and the hospital environment
(Yadav et al. 2020). Biofilms formed in medical devices
are responsible for the worsening of the patient’s clinical
condition. Currently, although many methods allow iden-
tification of biofilms in catheters and other devices, such
as electronic microscopy, biofilms on these materials are
not routinely investigated in clinical practice (Stoica et al.
2017).
Also, the conventional methods used in clinical labora-
tories for the identification of pathogens, which are based
on the use of a specific agar, the incubation of the sam-
ple, the isolation of the microorganisms and the enumer-
ation of viable cells, can only detect free-living bacteria
and cannot identify biofilm-forming microorganisms,
generating results that may not represent the clinical real-
ity of the patient (Mooney et al. 2018). Besides that, even
with the use of automated growth detection systems,
microbial identification takes approximately 5–7 days. In
the wait for the result, mainly with the presence of the
biofilm, these pathogens have already multiplied, worsen-
ing the patient’s clinical condition and forcing doctors to
start empirical therapy even without identifying the
pathogen, which can lead to errors in antibiotic therapy
and, in some cases, to the death of the patient
2 Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
N.B.S. Silva et al.
(Rajapaksha et al. 2019). To overcome this problem, new
and more sophisticated methodologies for the identifica-
tion of microorganisms in medical devices and clinical
samples must be implemented in clinical practice to make
the identification of microorganisms faster and to facili-
tate the routine investigation of biofilm formation in
clinical practice (Gaudreau et al. 2018; Ordonez et al.
2019).
The literature describes several methods of biofilm
detection, and the choice depends on the place of its
formation and the sample to be analysed; these methods
have greater specificity and sensitivity and are much less
time-consuming than other conventional techniques.
Biopsy is considered the most reliable way to detect bio-
films (Ordonez et al. 2019; Rajapaksha et al. 2019).
Biopsy samples are stained for the visualization of
microbial aggregates, the extracellular matrix of the bio-
film and cells of the immune system, and the larger the
size of the tissue and the number of biopsies collected,
the more sensitive and specific the result will be
(Kamaruzzaman et al. 2018). The viability method of
LIVE/DEAD uses green and red fluorescing markers and
can be used as a qualitative and quantitative technique
because it can differentiate healthy cells (stained green)
from damaged cells (stained red) in the biopsy
(Chevalier et al. 2017).
However, in many clinical situations, biopsies cannot
be collected or are not indicated, and other samples
could be sent to the laboratory, such as sputum, blood,
fluids and secretions (Percival 2017). In the laboratory,
analysis of these samples is difficult since the microorgan-
isms in the biofilm are adhered to each other and a sur-
face, forming a microbial aggregate. Therefore, sonication
is generally performed, using energy to agitate particles in
a solution, removing the aggregated and adhered bacteria
from biomaterial surfaces (Galli et al. 2018). After sonica-
tion, the aggregated microorganisms detach themselves
from the surface of the material and can be analysed by
several methods, as described below.
Polymerase chain reaction
This technique is used for the detection of biofilm-form-
ing pathogens directly in clinical samples. It is based on
the amplification of specific regions, providing high
specificity and sensitivity in the identification of genes
involved in biofilm formation, after the sonication pro-
cess (Rajapaksha et al. 2019). Some examples of devel-
oped PCR methods are real-time PCR, multiplex PCR
and reverse transcriptase PCR. These techniques can be
used to identify clinical samples that produce biofilms, as
in the study of Shahmoradi et al. (2019), who identified
biofilm-forming isolates of Staphylococcus aureus in urine,
N.B.S. Silva et al.
blood, sputum, cerebrospinal fluid, pleural fluid and
wound samples of hospitalized patients.
Moreover, PCR can be used to identify genes involved
in biofilm formation, as in the study of Klancnik et al.
(2015), who used the PCR-based method for the identifi-
cation of genes involved in the adhesion of different
strains of Listeria monocytogenes.
Fluorescence in situ hybridization
This method consists of the binding of short fluores-
cence-labelled oligonucleotides, which can bind to the
specific ribosomal RNA of the target organisms. It has
the advantage of allowing the analysis of the sample with-
out prior treatment, easily identifying microbial aggre-
gates; the samples are analysed via microscopy
(Frickmann et al. 2017). Bernardi et al., (2019) used the
fluorescence in situ hybridization (FISH) method and
confocal laser scanning microscopy (CLSM) in their
study with halitosis to visualize and quantify the biofilm
formed in the dorsum of the tongue, in which they iden-
tified Fusobacterium nucleatum and Streptococcus sp. bio-
film-forming strains. This method also has been tested in
bacterial vaginosis, identifying biofilm-forming bacteria in
vaginal samples, with greater specificity than for other
common methods (Machado et al. 2015).
Electronic microscopy
This approach allows the visualization of microbial aggre-
gates after sonication or FISH. Scanning electron micro-
scopy (SEM), transmission electron microscopy (TEM)
and CLSM are the most used tools in biofilm visualiza-
tion (Costa-Orlandi et al. 2017). The high resolution of
this microscopy allows analysis of all biofilm structures,
facilitating direct detection and visualization of the bio-
film where it was formed, such as in a catheter (Sugi-
moto et al. 2016; Schlafer and Meyer 2017).
Via SEM, samples can be fixed, dehydrated and
stained, but in all these processes, mainly in dehydration,
the morphology and structure of the biofilm can
be altered, which is a disadvantage of this technique
(Costa-Orlandi et al. 2017). The TEM is widely used for
biofilm architecture visualization and has an advantage
over SEM since the microbial aggregate is treated with
resin, which ensures greater stability of the extracellular
polymeric matrix of the biofilm. However, it has the dis-
advantage of not allowing visualization of the biofilm
topography (Mohmmed et al. 2017).
The CLSM guarantees the 3D visualization of the com-
plete biofilm architecture, besides allowing the identifica-
tion of macromolecules such as polysaccharides, proteins,
nucleic acids and lipids through the use of fluorescent
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
Diagnosis of biofilm infections
dyes, which mark the different components of the biofilm
as well as the extracellular matrix and spatial relationships
of the biofilm (Costa-orlandi et al. 2017).
The analysis of biofilms in vitro contributed to the
advancement of biofilm research; however, in most exist-
ing methodologies, biofilm is treated and analysed in a
different place from where it was formed, which can
damage its structure due to the lack of exact repro-
ducibility of the appropriate temperature, pH and avail-
ability of nutrients from the site of origin (Sugimoto
et al. 2016). To overcome this problem, a sophisticated
microscopic technique can be used in the detection of
the biofilm in situ, such as multiphoton laser scanning
microscopy, making it possible to analyse the biofilm at
the site where it was formed, such as catheters and
implants, preserving its structure and architecture. This
methodology is indicated for the acquisition of images of
autofluorescence materials, where the researcher can
obtain high-resolution images of the biofilm structure
without losing any important components of the biofilm
and without previous treatment of the sample (Neu and
Lawrence 2015).
Other types of microscopy are also used in biofilm
research, such as scanning transmission X-ray micro-
scopy, which is associated with X-ray absorption spec-
troscopy, Hoffman modulation contrast microscopy and
atomic force microscopy; however, they are associated
with high costs (Costa-Orlandi et al. 2017).
Even with the publication of the guidelines proposed
by the European Society for Clinical Microbiology and
Infectious Disease in 2014 and the current discussion,
until the present moment, the data about biofilm diagno-
sis and treatment are largely inconsistent, with little
information to support definitive conclusions for con-
tainment and prevention strategies. In addition, some
proposed methodologies are expensive or require signifi-
cant amounts of time and a large number of trained staff,
making them impracticable for clinical practice (Mooney
et al. 2018).
However, of all the methodologies mentioned above,
PCR and electron microscopy stand out due to their high
cost-benefit, short experimental time and high sensitivity
and specificity in the identification of microorganisms
(Rajapaksha et al. 2019). These techniques, associated
with the evaluation of signs and symptoms and clinical
samples of the patient, can modernize and improve the
way of diagnosing infections in the hospital, generating
faster results and using less laboratory inputs than con-
ventional techniques (Ordonez et al. 2019). In addition,
with the use of these methods, the doctor would be able
to distinguish infections caused by free-living or sessile
microorganisms, which will reflect in a better prognosis
for the patient, since the antimicrobial used would be
3
Diagnosis of biofilm infections
more specific and efficient for the infection, decreasing
the patient’s length of stay as well as the morbidity and
mortality rates in the unit (Høiby et al. 2015).
Antimicrobial susceptibility testing against
biofilms
Recently, there was a discussion in the literature, on the
implementation of a specific breakpoint value to assess
the bacterial susceptibility of biofilm-forming microor-
ganisms in laboratory diagnosis, but there was low appli-
cability; further studies and other pharmacological and
microbiological tests are needed for this value to be used
safely and effectively (Ruiz et al. 2019). Furthermore, it is
still early to determine a successful antibacterial regimen
against biofilms, as currently available indirect screening
tests may generate false negatives in the presence of
antimicrobial treatment, with the risk of relapse after
treatment discontinuation (Mooney et al. 2018).
Some studies recommend the use of combined antimi-
crobial therapy for the treatment of infections associated
with biofilms by Gram-negative bacteria through the use
of macrolides (erythromycin, clarithromycin and azithro-
mycin); however, determining a specific antibiotic ther-
apy for these infections is difficult due to the variety of
microorganisms that live in biofilms (Cepas et al. 2019).
This makes it difficult to determine a minimum inhibi-
tory concentration (MIC) of eradication for these sessile
microorganisms since to inhibit a biofilm, a drug at a
high concentration is required, and considering the side
effects and toxicity that antimicrobials have on human
cells, this type of treatment becomes impracticable
(Mooney et al. 2018).
Bacteria and fungi in biofilms are more resistant to
antimicrobials than free-living microorganisms, with min-
imum eradication concentrations of 10–1000 times higher
compared to planktonic bacteria (Mooney et al. 2018). In
biofilms, gene expression is altered, with negative regula-
tion of genes associated with glycolysis, carbon metabo-
lism and ribosomal proteins. In addition, the
transcription of genes encoding peptideoglycan, cell wall
synthesis and efflux pump activity are greater in sessile
cells than in planktonic cells (Castro et al. 2017; Nielsen
et al. 2019).
Vuotto et al. (2017), in their study with Klebsiella
pneumoniae urinary strains, demonstrated a correlation
between biofilm production and resistance profile, show-
ing that the samples that showed extensive resistance to
antimicrobials showed a greater ability to form biofilms
compared with sensitive samples. Neopane et al. (2018)
also observed that the S. aureus biofilm-forming samples
collected from wounds of hospitalized patients showed
higher levels of microbial resistance when compared to
4 Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
N.B.S. Silva et al.
nonforming ones. Furthermore, Mohammadi et al.
(2020) described a significant correlation between phos-
pholipase activity and biofilm production in Candida
albicans isolated from nails, discussing the importance of
biofilm formation to the reduction of azole drug penetra-
tion into the biofilm matrix and the increased expression
of gene resistance associated with antifungal resistance.
The relationship between biofilm production and
antibiotic resistance is not clear; however, a possible
explanation is that the plasmids that harbour antimicro-
bial resistance genes also harbour genes involved in the
production of biofilms (Vuotto et al. 2017), which would
explain why multiresistant microorganisms are also bio-
film producers. In addition, within biofilms, bacteria have
a cellular communication system called quorum-sensing,
which facilitates the exchange of genes within the com-
munity, including antimicrobial resistance genes (Osman
et al. 2019).
Therefore, several bacterial mechanisms may be
involved in microbial resistance within biofilms, such as
low antimicrobial penetration due to matrix presence,
antimicrobial and antifungal release strategies, altered
microbial multiplication rate and bacterial physiological
changes (Singh et al. 2017).
In 1999, the first strategy of the minimum biofilm
inhibitory concentration (MBIC) and the minimum bio-
film eradication concentration (MBEC) was created using
the Calgary biofilm device method. In this technique,
biofilm formation is tested with stakes that fit into the
wells of the microtiter plate, containing growth medium
and bacteria. Thus, microbes do not sediment and only
develop in the sessile form (Azeredo et al. 2017). Since
then, other procedures have been proposed, such as mod-
els based on bioreactors, flow cell systems and MBIC def-
initions evaluating biomass, but the application and
standardization of these procedures in the clinic are still
pending (Di Luca et al. 2017).
The MBIC of biofilm and the MBEC are used to
determine the efficacy of antimicrobials against biofilms
(Azeredo et al. 2017). These values are generally consider-
ably higher than the MIC of planktonic bacteria. Some
studies have been successful in developing drugs for the
treatment of catheter-related infections, taking into
account the MBIC and MBEC values of the biofilm, while
other clinical trials have failed to replicate these results
with other microorganisms from different sites. In addi-
tion, most microbial resistance studies of biofilms are
based on in vitro assays, questioning whether these tests
can be reproduced in vivo (Coenye et al. 2018).
Although several microbial susceptibility tests on bio-
films have already been described, so far, none has been
approved as a reference method in clinical microbiology,
generating an increasing interest in the development of
N.B.S. Silva et al. Diagnosis of biofilm infections

new specific sensitivity tests for sessile bacteria (Singh
et al. 2017).
Advances in scientific research: In vitro and
in vivo biofilm detection methods
Many in vitro and in vivo methods have been used for a
better understanding of the biology of biofilms and their
detection. Colorimetric assays are the most widely used
ones and commonly applied in studies related to the
development and susceptibility of biofilms to drugs and
the quantification of specific biofilm structures (Shukla
and Rao 2017). These methods, although requiring signif-
icant amounts of time and a high number of trained
staff, are important allies in identifying biofilm formation
and for a better understanding, mainly of the susceptibil-
ity of biofilms to anti-biofilms and antimicrobial com-
pounds, which can assist in the development of new
methods of diagnosis and treatment that can be useful in
clinical practice (Qu et al. 2017). The main in vitro and
in vivo tests used by the academic community are listed
in Table 1 and described below.
In vitro biofilm detection models
Congo red agar test
This agar allows the differentiation of the colonies in two
colours, with black indicating strong biofilm production
and red indicating no biofilm production (Lee et al.
2015). This is a quantitative test because it is based on a
subjective chromatic evaluation (Melo et al. 2013). Solati
et al. (2015) have used it to assess Staphylococcus epider-
midis produced by biofilm collected from the urine and
blood of patients with urinary tract infections and sep-
ticemia, respectively, in which they identified 55% of the
isolates with the greatest potential for biofilm formation;
most were collected from urine.
Tube biofilm formation test
This test consists of the use of plastic tubes containing
sample inoculum stained with 0
1% violet crystal. After
several washing processes with phosphate buffer saline
and fixation with sodium acetate, the tubes are inverted
in an adsorbent paper, and those with the presence of the
stained film on the bottom and walls of the tube, indicat-
ing the presence of biofilm, are being considered positive
(Halim et al. 2018).
Some studies have used this method to detect clinical
isolates producing biofilms; for example, Neopane et al.
(2018) have identified 30 S. aureus biofilm-forming iso-
lates collected from wounds of hospital-admitted patients,
while Hassan and Khider (2019) have evaluated the cor-
relation of the formation of biofilms in clinical samples
of Acinetobacter baumannii using the tube biofilm forma-
tion test and molecular tests. Despite being a low-cost
test, it is a qualitative and subjective test.

Table 1 Main in vitro and in vivo biofilm detection methods

Method Advantages Disadvantages References

In vitro biofilm detection

Congo red agar High accuracy in the detection of Low reproducibility Melo et al. (2013)
test Staphylococcus sp. biofilm producers
Tube biofilm Low cost Subjective reading Halim et al. (2018)
formation test
Microplate test Low cost Lack of standardization in the interpretation of Qu et al. (2017)
Several tests can be done simultaneously results
Crystal violet Low cost Low specificity Xu et al. (2016)
Simple technique
High replicability
Safranin Non-toxic dye Low replicability and sensitivity Stepanovıc et al. (2007);
Ommen et al. (2017)
XTT Simple technique High cost Costa-Orlandi et al.
High replicability Low sensitivity salt Retention by the pathogens (2017)
that can interfere with the result
In vivo biofilm detection
D. melanogaster High homologies between the Drosophila Preference for yeasts and Escherichia coli for Yamaguchi and Yoshida
and human genomes having small genomes (2018)
Easy to handle Does not have haemoglobin
Inexpensive to maintain
C. elegans Powerful methods for studying physiological Nematode culture standardization factors may Park et al. (2017)
processes interfere with its survival

Journal of Applied Microbiology © 2021 The Society for Applied Microbiology 5

Diagnosis of biofilm infections
Microplate test
This test uses microplates with flat bottom or U-shaped
wells, where all stages of biofilm formation occur within
these wells (Di Luca et al. 2017). It is a cheap technique
that requires few reagents, and several tests can be done
simultaneously. For the detection of biofilm production,
the wells with the samples are stained with specific dyes
that quantify the main structures of the biofilm. This part
of the experiment is the so-called ‘colorimetric test’ (Qu
et al. 2017). According to the absorbance result of each
isolate in these colorimetric tests, it is possible to classify
them into nonproducers, weak, moderate or strong pro-
ducers of biofilms. However, this classification is highly
subjective and can vary according to the species and the
cut-off value that the researcher uses as a reference
(Shukla and Rao 2017).
Crystal violet. In addition to the use of this dye in tube
tests, it can also be applied in microplate tests for an
assessment of the total biomass of the biofilm. It is a
cost-efficient substance, and the test is easy to interpret
and has high replicability (Shukla and Rao 2017). How-
ever, this dye has the disadvantage of staining the entire
structure of the biofilm, without specificity. Moreover,
after being deposited in the plate, the wells need to be
washed, culminating in the loss of important biofilm
components and structures (Xu et al. 2016). It is the
most used colorimetric test in the microplate test, making
it possible to quantify the entire biomass and matrix of
biofilms (Di luca et al. 2017).
In the literature, there are several studies that use crys-
tal violet in the identification and quantification of bio-
film production in bacteria and fungi (Xu et al. 2016;
Vuotto et al. 2017; Mohammadi et al. 2020).
Safranin. Safranin is a nontoxic dye, and its use is simi-
lar to that of crystal violet. However, it is less frequently
used in the quantification of biomass and results in lower
optical densities than crystal violet staining (Ommen
et al. 2017). Some authors, such as Babapour et al.
(2016) and Karami et al. (2020), have used safranin as a
dye in the microplate test for the identification of clinical
isolates of biofilm producers.
Some researchers believe that this dye specifically stains
the mucopolysaccharides of the extracellular matrix
because of their high affinity with these compounds.
Mucopolysaccharides are molecules produced by cells
within the biofilm that will compose the extracellular
matrix of the biofilm (Stepanovic et al. 2007). However,
not all biofilms have polysaccharides as a matrix
6 Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
N.B.S. Silva et al.
component, which is replaced by DNA and other com-
pounds. In addition, each microorganism has a type of
extracellular matrix which varies in its cellular localisa-
tion, chemical composition, nature and functions; many
are polyanionic, but others are neutral or polycationic
(Limoli et al. 2015).
The use of 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-
[(phenylamino) carbonyl 2H-tetrazolium hydroxide
(XTT). The tetrazolic salt XTT is used to evaluate the
number of viable cells and the metabolic activity of bio-
films. When microorganisms in the biofilm produce
metabolites, XTT changes their chemical conformation
and is transformed into formazan, which is seen in the
microtiter plate by a colour change from neutral to
orange (Costa-Orlandi et al. 2017). This compound is
most frequently used in detecting and evaluating the
metabolic activity of biofilms produced by fungi, as in
the study by Di Domenico et al. (2018), who detected 38
clinical isolates of Candida sp. biofilm producers, but it
can also be used for bacterial evaluation, as in the study
by Xu et al. (2016), who reported that 77% of S. aureus
isolates were biofilm-forming.
Some researchers use (3-[4,5-dimethylthiazol-2-yl]-
2,5iphenyltetrazolium bromide) MTT, which is a salt that
behaves in a similar way to XTT, albeit with a colour
change from neutral to purple. Comparing the two salts,
XTT has more advantages because after its reduction in
formazan, the wells can be read immediately, while MTT
requires other steps prior to reading, such as cell lysis
(Costa-Orlandi et al. 2017). Another disadvantage of the
MTT salt is the insolubility of formazan in water, making
it necessary to dissolve the precipitate before measuring
absorbance. In addition, reduced MTT can react with
some compounds, forming large intracellular deposits,
capable of damaging microbial cells during the test, pro-
ducing false positive or negative results (Grela et al.
2015).
However, XTT has some disadvantages, such as the
lack of a linear relationship between the number of
microorganisms present in the biofilm and the colorimet-
ric signal, salt retention by these pathogens in planktonic
and sessile form and a high cost (Alonso et al. 2017).
Additionally, XTT can react with some nanoparticles and
also be reduced by O2. Toxicity testing of engineered
nanomaterials including nano-TiO2 has generated a many
number of publications in the last several years (Wang
et al. 2011). Furthermore, problems regarding intraspecies
and interspecies variability have been reported, making
the use of these methods unfeasible in some susceptibility
tests for both planktonic cells and biofilms (Peeters et al.
2008).
N.B.S. Silva et al.
Detection of biofilms in vivo
Some researchers have used animal models to advance
the research and detection of biofilms in vivo. Due to
some difficulties encountered by the researchers in mam-
malian studies, including the approval of the Ethics Com-
mittee for such studies, other nonmammalian models are
being used, such as Drosophila melanogaster (McLaughlin
and Bratu 2015) and Caenorhabditis elegans (Park et al.
2017).
For example, Kamareddine et al. (2018) have used
D. melanogaster in their study of infection by Vibrio cho-
lerae, where they discovered that quorum-sensing, besides
being a cellular communication mechanism in biofilms,
also exercises control over the host’s metabolic pathways.
Another important study model for biofilms is C. elegans,
which is used for the evaluation of the progression of
intestinal infection caused by Salmonella sp., where it was
possible to identify a toxin that is secreted by these bacte-
ria in biofilm formation (Desai et al. 2019).
The advantages of such an approach are the ease of
handling and the lower costs. Moreover, the small size of
the models makes it easy to keep them in microtiter
plates, thus facilitating the high-performance screening of
biofilm formation and making them important tools in
understanding the pathogen-host relationship (Costa-
Orlandi et al. 2017).
Matrix-assisted laser desorption ionization time-
of-flight mass spectrometry (MALDI-TOF MS): Is it
the diagnostic method of the future?
In recent years, mass spectrophotometry has been used
by some clinical laboratories for the identification of bac-
teria and fungi. It is a method that compares the micro-
bial protein profile, obtained from a database of reference
mass spectra, for the identification of genera, species and
subspecies. Microbial identification is performed in 60 s
and has been used in the diagnosis of several infectious
diseases around the world (Singhal et al. 2015). Cur-
rently, this methodology is the target of researchers and
applied biofilm studies, since MALDI-TOF allows the dif-
ferentiation of planktonic cells and biofilms due to pro-
files of different mass spectra (Caputo et al. 2018). The
method was tested on different species of Gram-positive
and negative bacteria and fungi (Kubesova et al. 2012;
Pereira et al. 2015; Caputo et al. 2018), showing that it is
possible, through MALDI-TOF, to differentiate microor-
ganisms that form weak or strong biofilms, based on
their molecular mass size.
However, due to several problems and limitations of
this technique, MALDI-TOF is far from being the gold
standard for identifying biofilm formation. A common
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
Diagnosis of biofilm infections
problem encountered by researchers is that the same
microorganisms may present different mass spectra when
analysed repeatedly (Singhal et al. 2015). Another limita-
tion of the technique is the difficulty in differentiating
microbial samples with a high degree of similarity due to
the several similar compounds included in their cells. In
addition, other intracellular compounds that vary within
each microorganism may not be detected by the equip-
ment; especially if they belong to low- or high-mass pro-
teins, which are generally not ionized by the apparatus
(Mlynarikova et al. 2016). However, according to Gau-
dreau et al. (2018), sample preparation for analysis is cru-
cial for the success of the test, and when the sample is
transferred directly to the analysis, together with the pres-
ence of formic acid and the matrix, the chances of inter-
ference are lower compared to other types of preparation
with acetonitrile, water and ethanol.
New studies are necessary to prove the efficacy of this
methodology in the detection of biofilms, since it pre-
sents limitations and low reproducibility. While MALDI-
TOF is not yet fully effective in the detection of biofilms,
conventional in vitro and in vivo techniques are still most
frequently used in research labs (Singhal et al. 2015).
Omics analysis: The role of proteomic,
transcriptomic and metabolomic analyses in the
diagnosis of biofilms
The traditional methods of microbiological diagnosis in
clinical laboratories include the growth and isolation of
the microorganism, the detection of antibodies or anti-
gens in serological tests and, in some laboratories, genetic
identification (DNA or RNA) through the PCR technique
(Ordonez et al. 2019). While most molecular tests target
only a limited number of pathogens using specific pri-
mers, omics analysis allows to analyse the entire micro-
biome of the pathogen and to identify specific proteins
and metabolites produced by pathogens (Otto et al.
2012). These technologies are also being developed to
detect differences in the transcription, translation and
metabolism of microorganisms in biofilms, describing
important information of these communities, such as the
influence of environmental factors on biofilm adhesion
and formation as well as physiological and mutational
factors (Chiu and Miller 2019). Table 2 shows the main
methods used in the omics analysis as well as the samples
that can be evaluated and the infections that can be diag-
nosed.
The study of proteomics in biofilms allows the identifi-
cation of specific proteins that are produced in this com-
munity, as well as the distribution, posttranslational
modifications, structure, function and quantity of these
proteins (Washio and Takahashi 2016). The success of
7
Diagnosis of biofilm infections
Table 2 Omics analysis in the clinical diagnosis of biofilms
Detection methods Clinical samples
Transcriptomic
The express sequence tag (EST) Blood
Serial Gene Expression Analysis (SAGE) Peripheral blood mononuclear cel
DNA microarrays Several body fluids and tissues
RNA-Seq (4sU-Seq, RIP-Seq, (Riboseq)) Cerebrospinal fluid
Proteomic
2D gels (DIGE) Blood
Gel free (iTRAQ, TMT)
Spiked-in peptides Secretions
Label-free quantitation Cell-free body fluids
Metabolomic
Capillary electrophoresis (CE) Blood
Fluids
Time-of-flight mass spectrometer Stool sample
(TOF-MS)
CE-TOFMS Cerebrospinal fluid
proteomics studies is due to the invention of the 2DE
gel, which is applied in orthogonal separation to classify
species according to their molecular weight, where sam-
ples are stained and identified using software (Otto et al.
2012). Despite being a technique similar to MALDI-TOF,
it is more sensitive and can differentiate nonstressed from
stressed cells, cellular mutations and different time points
from the same organism. The combination of proteomics
analysis with the use of 2D gel and MALDI-TOF can gen-
erate more sensitive and specific data, revealing cases of
multiple proteins found in the same place and those with
low mass, reducing false values and improving the disad-
vantages of MALDI-TOF (Otto et al. 2012; Singhal et al.
2015).
Erdmann et al. (2019) used proteomics analysis and
identified 1021 different proteins in the biofilms pro-
duced by 27 samples of Pseudomonas aeruginosa and a
protein for mRNA that was common among species,
even under different conditions, suggesting a posttran-
scriptional pattern. If researchers were able to identify
proteins common in most biofilms produced, it would
be possible, through the methods mentioned above, to
reduce the time needed for the identification of
microorganisms, facilitating our understanding of the
main regulatory pathways and adaptation strategies for
bacterial biofilms, which can be useful in the develop-
ment of new treatment strategies for these biofilms
(Chiu and Miller 2019).
Transcriptomics analysis allows identifying an RNA
molecule in a live cell. In the 1990s, several methodolo-
gies were created that allowed sequencing specific RNA
8 Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
N.B.S. Silva et al.
Clinical indication Reference
Sepsis Hrdlickova et al. (2017)
Urinary Tract Infection
Multiplexed pathogen detection
Meningitis
Sepsis Otto et al. (2012)
Urinary Tract Infection
Respiratory infections
Sepsis Chiu and Miller (2019)
Washio and Takahashi
(2016)
Infections by Clostridium difficile
Meningitis
molecules on a large scale, such as the express sequence
tag (EST) method, serial gene expression analysis (SAGE)
and DNA microarrays; however, they have numerous dis-
advantages, such as high costs, and demand for informa-
tion on the sample genome and transcriptome, impeding
discoveries (Hrdlickova et al. 2017). The method of
choice for studying gene expression and identifying new
species of RNA is RNA-Seq, as it offers less background
noise and greater detection than previous methodologies
(Hrdlickova et al. 2017). The transcriptomic approach is
of great interest in biofilm research, as it allows the detec-
tion of specific genes in the formation of biofilms and
new mechanisms of survival and antimicrobial resistance
(Chiu and Miller 2019). For example, Kean et al. (2018)
used this approach to investigate the mechanisms of bio-
film formation and resistance in Candida auris, a recently
discovered yeast.
The metabolic analysis is based on the identification
and quantification of metabolite features in living cells,
including gene transcription, mRNA translation, protein
analysis and enzymatic reactions (Zhang and Powers
2012). In the 2000s, several devices with a metabolic
approach were used, such as capillary electrophoresis and
time-of-flight mass spectrometer (TOF MS), which iden-
tify metabolites according to their ionic charge and mass
spectrometry, respectively. By connecting these two
devices, these techniques can separate small ionic mole-
cules and accurately measure their masses; they have been
widely used since then. The metabolic approach provides
data that allow the diagnosis of a disease as well as its
progress (Washio and Takahashi 2016).
N.B.S. Silva et al.
This allows, for example, the identification of chemical
signalling molecules, so-called ‘autoinducers’, responsible
for cellular communication within biofilms. Autoinducers
are targets of promising drugs for biofilms since these
molecules are not present in the human organism and
can therefore be used as biofilm markers (Mooney et al.
2018). The molecular structure of a biofilm can positively
impact the biology of systems and, consequently, the
development of new diagnostic and/or therapeutic tools
(Zhang and Powers 2012).
Advantages and disadvantages of omic analysis
The new omic approaches allow an evaluation of several
biological units of biofilms, such as gene expression, pro-
teins and metabolites. This generates highly relevant data
for a better understanding of biofilms, allowing the dis-
covery of new biomarkers for various diseases and the
development of new methods of diagnostics and therapies
(Chiu and Miller 2019). The application of these omic
methods in the health area can generate opportunities for
clinical interventions, as in the study by Nascimento
et al. (2017), who through omic analyses identified new
oral Streptococcus and their phenotypic characterization
revealed promising candidates for probiotic therapy. To
investigate complex multispecies biofilms, the use of omic
tools is the promise of a breakthrough regarding the
understanding of bacterial interaction and physiology
within biofilms.
However, although the transcriptomic, proteomic and
metabolomic analysis are complementary methods to
investigate the cellular physiology of biofilms, these omic
methods were not designed to be evaluated together; that
is, it is not possible to validate or compare one data with
the other (Azeredo et al. 2017). Also, biofilm is by defini-
tion heterogeneous, varying according to its location,
stage of development and physiology of microbial cells.
In the omic analysis, the data result from a cell that will
represent the entire population, consequently, a subpopu-
lation can be neglected or be different from the cell col-
lected for analysis.
Therefore, the results of the omic analysis should be
analysed with caution, considering that the results
obtained are averages with margins of error (Jamal
et al. 2018). However, flow cell cameras can work
around this problem, as microbial cells grow statically.
Another solution is the laser capture microdissection
microscopy as it allows the comparison and identifica-
tion of the physiology of cells and their special distribu-
tion within the biofilm, for example, the differences
between cells that reside on the periphery of biofilms
with those that live in the superficial regions. The main
challenge that a remains unsolved is how much biomass
Journal of Applied Microbiology © 2021 The Society for Applied Microbiology
Diagnosis of biofilm infections
is needed to perform molecular analyses (Azeredo et al.
2017).
Another question that remains under investigation con-
cerns the reference to compare the omic data obtained.
Often a planktonic cell is used as a control, but it may not
be the most appropriate, since the biofilm has several
stages of development and any attempt at correlation
would be incorrect. The best choice for the investigation
of biofilm physiology remains to compare a wild-type
strain with an isogenic mutant (Zhang and Powers 2012).
The biofilm matrix can also be a problem for proteomic
analysis since it can make access to different intracellular
membrane and parietal subproteomes difficult (Washio
and Takahashi 2016). Also, the analysis of exoproteoma,
that is, proteins that are secreted into the extracellular
environment, is also a challenge (Desvaux et al. 2009). In
addition, omic analysis requires qualified technical exper-
tise and is expensive.
Despite the challenges that these techniques still con-
front, the development of omic technologies is essential
for a better understanding of the development and evolu-
tion of biofilms, allowing the creation of new methods of
diagnosis and treatments in the health area (Chiu and
Miller 2019).
Conclusions
Despite all advances in research, biofilm-producing
microorganisms remain a serious public health problem,
often neglected in various health areas. Biofilm con-
tributes to increased microbial resistance, making treat-
ment more difficult and worsening the clinical conditions
of patients, contributing to the increase in morbidity and
mortality rates. The diagnosis of biofilms is challenging,
since the current screening methodologies performed in
the clinic, such as susceptibility tests, detect only plank-
tonic colonies, not representing the clinical reality of the
patient.
Also, even with several biofilm detection methods
described in the literature, there is currently no routine
test or protocol established for this purpose in the clinic.
Omics analysis is a promising form of diagnosis in clini-
cal laboratories since it allows the identification of patho-
gens in less time than needed for conventional
techniques, and it is possible to analyse the entire patho-
gen microbiome, including an identification of produc-
tion of proteins and metabolites, which allows a better
understanding of its pathogenesis and a more specific
identification.
More studies must therefore be developed to facilitate
the development of efficient and reliable protocols for
rapid biofilm identification, ensuring greater chances of
success in infection control in clinical practice.
9
Diagnosis of biofilm infections
Conflicts of Interest
No conflicts of interest are declared.
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