MATERI 2

Biofilm Disease

Pemateri: Prof.dr. Titik Nuryastuti, Sp.MK, Ph.D.

Biofilms disease:
How to diagnose

Titik Nuryastuti
Dept of Microbiology, Fac of Medicine UGM
OUTLINE
Microbiological
Pathogenesis features

Clinical
Diagnosis BAI
Presentation
Biofilm..clinical history
The term biofilm, originally used in technical and environmental microbiology, was introduced into
medicine in 1982 by Costerton
Biofilm lifecycle

(Von Eiff, 2002) (Magana, 2018)

Pathogenesis
Pathogenesis
BIOFILMS may form on both biotic and abiotic surfaces.
• Bacterial colonization of these devices can occur rapidly (within 24 h)
• Enhanced by host-produced conditioning films (such as platelets, plasma, and tissue
proteins)
• medical device is placed : becomes a site that host cells and microbes compete to
reach
• colonization of the material surface of the medical device, followed by the complex
metamorphosis of the microorganism that results in the formation of a biofilm
• microbial source can be acquired either from
• indigenous microbiota
• exogenous sources
Origin of microbes

(Bjarnsholt T, 2013)
Port de entry of biofilm disease

direct inoculation into microbial spread through

the surgical wound blood from a distant
during surgery or focus of infection
immediately thereafter (hematogenous
(perioperative infection) infection)

due to penetrating
direct spreading from an trauma (contiguous
adjacent infectious focus infection)
Epidemiology ....
• Once a device has been implanted, host tissue cells and pathogenic
bacteria compete to gain a presence on the surface of the device.
• rates of biofilm-related device infection: (Pervical et al, 2015)
• 2% for joint prostheses and breast implants
• 4% for mechanical heart valves, pacemakers and defibrillators
• 10% for ventricular shunts
• 40% for ventricular assist devices
• most nosocomial bloodstream infections are associated with intravascular
devices
• Early infections occur within 2 weeks of implantation and are associated with
intraoperative wound contamination;
• late-onset infections are often occult, prolonging recognition of disease by weeks,
months, and in some cases, years
Clinical Presentation..
(Pozo, 2018)
Relevance of biofilm disease
in clinical practice
• device-related biofilm disease
• non-device related chronic biofilm disease
• biofilm-related device malfunction.
Device-related biofilm disease
• device inserted or implanted
material serving as the surface for
attachment.
• amenable to easy removal
• The risk of removal may be so great
that true eradication of the infection
may not be possible without risking
the patient’s life.
• In such cases, clinicians may
choose to suppress the infection
with chronic antimicrobial therapy.
• relapse is highly probable if
antimicrobial suppression be
discontinued for any reason
Non-device related chronic biofilm disease
• having aqueous
microenvironments with surfaces
including devitalized bone (eg,
mastoiditis and osteomyelitis)
• accumulated inflammatory tissue (eg,
sinusitis, cystic fibrosis-related
bronchitis),
• mineral deposits: cholelithiasis and
renal stones
• These surfaces provide protected
sites across which nutrients flow
• Eradication of the infection is
dependent on removal of the
colonized surface
(Pozo, 2018)
HAIs and Typical biofilm infection

(Pevical et al, 2015)

(Nuryastuti T, et al, 2018)
Biofilm-related device malfunction
• Biofilm microorganims growing of this foreign body
reaction are now known to lead to degradation of
the biomaterials with subsequent clinical device
failure.
• Depending on the balance of the interactions
between the host, implant, microorganisms and
their byproducts, different clinical presentations
may vary :
• Implant malfunction
• light pain
• slight soft tissue contracture
• functional impairment
• with negative infection/inflammatory markers.
• Diagnosis of subclinical infection often requires
prolonged cultures, specific microbiological
laboratory procedures (sonication) and eventually
genomic investigations for detection of biofilm
pathogens

(Pozo, 2017)
How to diagnose biofilm disease
Diagnosis of biofilm disease
Clinical evidences of biofilm infection
• typical biofilm infection is usually a
chronic, persistent infection with
intermittent exacerbations/relaps
• antibiotic treatments could be
helpful to control the acute
exacerbations, but difficult to
eradicate the infection.
Microbiological examinations
• Be aware about sample collection, microbial
cultivation, identification method
• Device related : at least 4–5 pieces of tissue biopsy
from different sites next to the prosthesis
suspected infection
• The prostheses, catheters or stents and other
foreign bodies taken out from patients due to
suspicion of biofilm infections should be sent for
microbiological examinations.
• culture-negative samples, if the patients are highly
suspected PCR 16s rRNA might be helpful
• Gram staining, Giemsa
• FISH
(Hall-Stoodley , et al, 2012)
General features of clinical and
laboratory indications for diagnosis of biofilm
infections
• Clinical signs of infection: low-grade inflammatory respons
• History of biofilm-predisposing condition (e.g. implanted medical device, cystic fibrosis)
• Persisting infection lasting >7 days or resistance to the antibiotics used
• Failure of antibiotic treatment and recurrence of the infection (same organism is responsible on
multiple time points)
• Evidence of systemic infection resolve with antibiotic therapy, recur after therapy has ceased.
• Microbiological diagnostics:
• fluid/tissue samples revealing the presence of microbial aggregates and biofilm structure co-localized with
inflammatory cells
• Positive culture/non-culture-based techniques (PCR) of fluid or tissue sample
• Presence of mucoid colonies or small colony variants which may indicate antibiotic recalcitrance
• FISH positive results for known pathogen showing aggregated microoganisms
• Specific immune response to identified microorganism

Parsek, 2003; Stoodley, 2009, Høiby N, et al, 2014

(Hong Wu, 2015)
(Hong Wu, 2015)
Which type of samples to detect biofilm
infections..?
• Lower airways secretion (sputum, BAL, endolaryngeal suction
(from small children), induced sputum
• Chronic wound infection : Biopsy tissues, soft tissue sample from
the base of the debrided wound
• Infections related to an orthopedic device: synovial fluid, biopsies
from representative peri-implant tissue, removal of the
device/prosthesis or modular parts of it (e.g. inlay, screws)

Høiby N, et al, 2014

Cont..Sample collection..

• Endotracheal tube biofilm (VAP): Mucus and biofilm retrieved from the inner
surface of the ETT
• Patients with vascular catheters: catheter tip (3 to 4 cm distal)
o If catheter-related infection is suspected : paired blood cultures from the vascular catheter
and peripheral blood taken simultaneously.
• Patients with indwelling urinary catheters or urethral stents:urine samples from
the catheter, removed catheters

To obtain biofim cell :

• rolling (the Maki method), scraping, whirly-mixing
• vortexing and/or sonication before culturing
Which methods should be used to detect biofilms
in the samples?
• Microscopy: presence of leucocytes, microorganisms present in microbial aggregates embedded in an
apparently self-produced matrix:
• Gram stain, CLSM, SEM, fluorescence in situ hybridization (FISH) probes
• Culturing on enrichment agar : Persister cell manifests as SCVs
• isolation of SCVs (small colony variants) from biofilm-associated infections
• differ from the normal phenotype : small colony size <1 mm, pinpoint or ‘fried egg’ colonies
• reduced growth rate, pigmentation and haemolysis, more resistant to aminoglycosides and cell-
wall inhibitors, and have a tendency to persist
• Persisters are small subpopulations of bacteria that survive lethal concentrations of antibiotics without
any specific resistance mechanisms
• CFUs do not always directly correlate with cell membrane permeability and enzyme activity, bacteria in
biofilms may be membrane compromised and nonculturable but still viable under stressful, nutrient limiting
conditions
• PCR techniques cannot discriminate between planktonic and biofilm-growing bacteria, however
(Kahl et al, 2016)
Biofilm development
Biofilm Formation

2 hours 4 hours

8 hours 24 hours
Olson ME, Ruseka I, Costerton JW. Colonization of n-butyl-2-cyanoacrylate tissue adhesive by Staphylococcus epidermidis. Journal of Biomedical Materials
Research 1988;22:485-495.
Pathogens that have been studied for the formation
of biofilms
 S. aureus  H. Influenzae
 S. epidermidis  Vibrio sp
 Streptococcus mutans  S. pneumonia
 Salmonella typhi  Klebsiella pneumonia
 Enterococcus sp  Acinetobacter spp
 Pseudomonas  Burkholderia sp
aeruginosa  Non tuberculous
 E. Coli mycobacterium
 Fungi : Candida spp,
Aspergilus spp
Analytical techniques for monitoring
biofilms
 Indirectdection of organisms by analysis of waste
and/or metabolism by products (GC/MS, HPLC, etc.)
 Direct detection of organisms
 Tube method
 Culturing on specific media : CRA
 Microtiter plate method (MTP)
 Direct observation by Microscopy techniques : CLSM, SEM,
TEM, FISH
 Biofim related gene
Methods to detect biofilm-forming microorganism
in vitro
Tube method (TM)
A qualitative assessment of
biofilm formation
 Trypton Soya Broth
 stained with crystal
violet (0.1%).
 positive when a visible
film lined the wall and
bottom of the tube.
 (-) observer dependent
and highly qualitative

(Ira P, et al, 2013)

Congo Red Agar (CRA)
 The medium was composed of BHI (37 gr/ L), sucrose (50 gr/L), Bacto agar
(10 g2/L) and Congo red stain (0.8 gr/L). congo red is a well known
Amyloid binding dye and its is reported that S. aureus producing Amyloid
for biofilm formation
 Positive result was indicated by black colonies with a dry crystalline
consistency
 Weak slime producers usually remained pink, though occasional darkening
at the centers of colonies
Atshan et al, 2012
 CRA method showed very little correlation with
either of tube or tissue culture plate method and the
parameters of sensitivity (7.6 %), specificity
(97.2%) and accuracy (51.3%) were very low
 Screening on CRA does not correlate well with
corresponding methods for detecting biofilm
formation in staphylococci
 Only can not be applied for staphylococci (produce
amyloid matrix)
(Pantanella F, et al 2013)
Tissue Culture Plate method (TCP) and
Crystal Violet (CV) staining
 Tissue culture plate method is most widely used and
was considered as standard test for detection of
biofilm formation. It was aimed to quantify the
biofilm more objectively
 recommended as a general screening method for
detection of biofilm producing bacteria: sensitive,
accurate and reproducible
 a quantitative model to study the adherence of
microorganism on biomedical devices
Crystal violet staining / XTT assay
Microtitre plate method
Biofilm formation takes place as a ring around the
well. After rinsing of wells to remove planktonic cells,
the biofilm can be stained with crystal violet and
dissolved in acetone–ethanol for quantification of the
biomass by measuring the OD :
(+) ease, rapidity and reproducibility of the method.
( - ) the absence of a relationship between biomass
and biofilm viability, as crystal violet stains dead and
viable cells equally

(Pantanella F, 2014)
For Staphylococci

Mathur, et al, 2006

Can be applied for all microorganism
Hassan A, et al, 2011
 Crystal violet staining

Tube method

Microtiter plate method

Mathur T, et al, 2006

 Confocal Laser Scanning Microscopy (CLSM)

 to scan a thick biological sample, e.g. a microbial biofilm, by processing images,

line by line, in X, Y and Z axes, stained with specific fluorescent dye
 biofilm formed on various surfaces, even transparent or non transparent surfaces
such as silicone, polyethylene or stainlsess steel, polymethyl metacrylat
(-) semiquantitative investigation and that only few fluorescent stains can be
employed

Projected top-views of 24 h biofilm of S. epidermidis grown on PE (A), PMMA (B), and SS (C) obtained using
CLSM after staining with Baclight dead/live stain (Nuryastuti T. et al., 2011)
Comparison of MTP method and CLSM images

(Nuryastuti T, et al, 2008)

Scanning Electron Microscope (SEM)

 to observe the morphology of bacteria adhered on

a material surfaces, the morphology of the material
surface, and the relationships between them.
 provides also information about the morphology of
biofilm cell and presence of EPS.
 Biofilm morphology and mass are important characteristics
that control the kinetics of substrate removal by biofilms
SEM images of candida biofilm cell treated by Phenantroline

Control -
PCR and Real time PCR to detect Biofim
related gene
 Application of Molecular technique often destroys
biofilm morphology or architecture due to the
DNA/RNA extraction process.
 Real Time PCR showed a meaningful tool to compare
genes expression involved in biofilm formation of
clinical isolates under different environmental
exposure. Using these data, the regulation of biofilm
formation can be studied more clearly.
Biofilm formation and ica expression increased by the exposure of subMIC of
some antibiotics and cinnamon oil

(Rachid et al, 2007, Nuryastuti T, et al, 2010)

Antibiotic tolerance...or...Antibiotic resistance

 Related to the presence of persister cell

 Persisters are not mutants; phenotypic variants of
actively dividing cells, reversible, non-hereditary
 Persisters are non-growing dormant cells, explains
their highly tolerance to bactericidal antibiotics
 All of the biofilm-forming pathogens examined so far
form persisters but the mechanisms underlying the
formation of persisters is still largely unknown
 planktonic persisters are cleared by the host immune
response, biofilm persisters are shielded from host
defence, and may cause a relapse of infection
Fig 1. Typcal Biofilm Infection
(Haiby et al, 2014)
FIG.2. Biofilm causing tissue infection. Biofilms of P. aeruginosa from sputum of cystic fibrosis patients.
Gram-staining (a-e), PNA-FISH staining with a P. aeruginosa specific probe (f, g). The bacteria and the
matrix are visible. A diversity of shapes of the biofilms are seen, a: with surrounding polymorphonuclear
leukocytes (arrows), b & c: with a few leukocytes within the alginate matrix, d: with channel-like holes
(arrow), e: with liberated planktonic bakteria (arrow)

(Bjarnsholt et al ,2009
Crystal violet staining / XTT assay
Microtitre plate method
Biofilm formation takes place as a ring around the
well. After rinsing of wells to remove planktonic cells,
the biofilm can be stained with crystal violet and
dissolved in acetone–ethanol for quantification of the
biomass by measuring the OD :
(+) ease, rapidity and reproducibility of the method.
( - ) the absence of a relationship between biomass
and biofilm viability, as crystal violet stains dead and
viable cells equally

(Pantanella F, 2014)
Which methods should be used to detect biofilms
in the samples?
• FISH and CLSM is another sensitive and specific approach
• well suited to the study of complex tissue samples and evaluation of
the presence of microbial aggregates
• FISH relies on hybridization of a fluorescently labeled probe to the
16S or 23S ribosomal RNA (bacteria) or the 18S or 26S (fungal/
protozoan pathogens)
• Identify whether the bacteria present are aggregated
• Indicate a polymicrobial nature of a biofilm
• indicate the extent of biofilm on a surface that CFU may vastly underestimate
• to show biofilm EPS that may comprise a greater part of the biofilm than cells
alone
(Hochstim et al,2010)
FISH CLSM
Strep endocarditis Infected suture
FISH

CF
Chronic wound

(Gescher,2008)
 Confocal Laser Scanning Microscopy (CLSM)

 to scan a thick biological sample, e.g. a microbial biofilm, by processing images,

line by line, in X, Y and Z axes, stained with specific fluorescent dye
 biofilm formed on various surfaces, even transparent or non transparent surfaces
such as silicone, polyethylene or stainlsess steel, polymethyl metacrylat
(-) semiquantitative investigation and that only few fluorescent stains can be
employed

Projected top-views of 24 h biofilm of S. epidermidis grown on PE (A), PMMA (B), and SS (C) obtained using
CLSM after staining with Baclight dead/live stain (Nuryastuti T. et al., 2011)
Comparison of MTP method and CLSM images

(Nuryastuti T, et al, 2008)

Treatment challenges
Treatment for biofilm disease
• Current antimicrobial therapies do not match to reliably eradicate infections involving biofilms
• there is microenvironments where antimicrobials have diminished effectiveness.
• treatment failures and relapsing symptoms despite seemingly appropriate antimicrobial treatment
• whenever possible, identifying and removing biofilms (or the device on which they form) is the key to
successful care.
• When removal is not possible, chronic antimicrobial suppression may be the only safe option.
• Suppressive antimicrobial treatment : narrow spectrum ab to which the pathogen is confirmed to be susceptible in routine
(ie , planktonic) testing.
• Although the primary site of infection will not be eradicated, appropriate blood levels of a targeted antimicrobial are used to
kill any viable organisms released from the biofilm, and to prevent systemic infection and seeding of secondary sites.
• combination of antibiotics with different killing mechanisms can sometimes eliminate the infection
• Administration of antibiotic: local (base on site of infection) and systemic
• high dosages of antibiotics under the safe range of renal and hepatic functions are suggested
• proper duration of antibiotic treatment is also important
• Appropriate debridement when indicated
• Methods for measuring antimicrobial susceptibility of organisms in the biofilm mode of growth
Manajemen approach
• to trigger conversion from biofilm to planktonic growth (eg , for
existing infections not amenable to removal) by identification of
synthetic molecular signals
• to prevent biofilm formation
• inhibiting quorum-sensing by blocking the appropriate receptors (eg , on
implantable devices)
• Antimicrobial flushes or locks that are used for vascular access devices may
have an impact on biofilms within the device.
(Pozzo, 2018)
 Ex. antibiofilm research of natural compound

• Biofilm formation of S. epi increased by the exposure of subMIC of cinnamon oil

• Cinnamon oil is able to detach and kill existing biofilms of S.epidermidis,
Chloroprocta sp. maggot filtrates, containing gelatinase
has an antibacterial activity
• increase the reduction of the extracellular matrix and
• decrease the viability of embedded S.
epidermidis biofilm
Ex. antibiofilm research of synthetic compound
• (1)-N-2-methoxybenzyl-1,10-phenanthrolinium showed
disruption of C. albicans biofilms
• It reduce the density of matrix C. albicans biofilm
 Take home messages
• Better distinguish Biofilm associated infection from acute planktonic
ones
• Obtain appropriate clinical samples
• Provide focus for the development of routine clinical test of biofilm
associated infection