A report on the approaches to limit the influence of bacterial biofilms in implanted medical devices

1. COVER SHEET
Student Name Malavika Sankararaman
Student Number 18200405
Assessment Title
Microbial interactions with implanted medical devices -
Approach to limit the influence of bacterial biofilms in medical devices
Module Code BIOC40130
Module Title Medical Device Technology
Module Coordinator Prof. Cormac Murphy and Prof. David FitzPatrick
Date Submitted 18-03-2019
Date Received
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Date 18-03-2019

2. MICROBIAL INTERACTIONS WITH
IMPLANTED MEDICAL DEVICES:
Approaches to limit the influence of bacterial
biofilms in medical devices
Abstract
Microbial infections related to medical implants constitute a large proportion of
nosocomial infections. Depending on the implant type, surgical site and indwelling
time period, the severity of these infections varies. Plaques of bacterial commu-
nities, termed as biofilms which form on the device surface, account for >80 % of
these microbial infections. These biofilms are a major threat, since they protect
the bacterial cells, by neutralizing the host immune response, preventing antibi-
otics from reaching the microbes and by dispersing the pathogens to other body
parts, thus causing adverse tissue damage, device dysfunction and chronic infec-
tions. These microbial communities are a nightmare to the healthcare sector, due
to the difficulty in treating and preventing them, because of their high tolerance,
varying phenotype and resistant characteristics. Conventional treatment methods
include antibiotics administration, device removal or implant replacement, all of
which are expensive and are less effective, because of the recurrence of bacteria. In
this essay, we explore the mechanism of biofilm formation, adverse effects caused
by them, current treatment methods and also discuss the novel approaches such as
vaccination and antibiofilm coating, that are being developed to limit the influence
of bacterial biofilms in implantable medical devices.
1 Introduction
Healthcare associated infections (HCAI), also known as nosocomial infections, occur in
patients, who are undergoing or have underwent a healthcare service and are caused
by bacteria, fungi, prions, viruses, and parasites. Reports released by several healthcare
bodies have confirmed that, about 60% - 70% of these HAIs are associated with implants,
among which, the incidence rate is high for central venous catheters, heart valves, and
urinary tract catheters [Bryers (2008)]. Medical implants are widely utilized in cardio-
vascular, orthopedic, and neurological areas, to replace, support or improve a biological
structure within the body. However, the functionality of these life-saving medical devices
is at a stake, because of the prevalence of these microbial infections, which occur due to
unhygienic practices/environment.
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3. Figure 1: Examples of implantable medical devices
Currently, the demand for implants are rapidly growing, mainly due to the advance-
ments in technology, prevalence of diseases, and increase in awareness. The global implant
market is dominated by orthopaedic and cardio vascular implants, due to the ageing pop-
ulation and has been estimated to reach a size of US$140.67 billion by 2023.
Figure 2: Graph illustrated using the data obtained from the report released by WHO
Every year, 5 million device implantations are being carried out in the regions of U.S.A
and 2 million in Europe, for which the infection rate varies from 1% to 40% depending
on the surgical site and implant duration. Regardless of the rising demand for medical
implants, and the cruciality it plays in human lives, most of these devices are vulnerable
to infections, in which, >80% occur due to the clustering of bacteria within a matrix
layer, called as biofilms [Khatoon et al. (2018)]. These infections pose a serious threat to
the patients, as they extend hospital stay, develop chronic infections, spread antibiotic
resistance, and cause death.
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4. Figure 3: The magnitude of problem of device associated infections
2 Literature Review
The common microorganisms behind implant infections, as mentioned by M Donlan
(2001), include both gram negative and gram positive bacteria, such as Staphylococcus
aureus, and Staphylococcus epidermidis, which accounts for 50-70% of catheter infec-
tions, and 40-50% of heart valve infections, as reported by Chen et al. (2013). VanEpps
& Younger (2016) has reported that there is a 5% mortality rate for low to medium risk
implants, such as Foley catheters and dental implants, while death rate due to the in-
fections associated with mechanical heart valves, pacemakers and other high risk devices
are more than 25%. Also, according to Saint (2000) and Lynch & T Robertson (2008),
the treatment costs of biofilms, have been estimated to range from $2836 for low risk
devices such as catheters, $15,000 for higher risk implants like orthopaedic devices, and
more than $50,000 for infected mechanical heart valves. This mortality rate and high
costs are due to the difficulty in treating biofilms since they are highly resistant towards
therapies, as quoted by Percival et al. (2015).
This highlights the urgent need to develop effective methods to minimise the effect of
biofilms on implants, which will be further discussed in this essay, along with a review
on upcoming methodologies.
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5. Figure 4: Indwelling medical devices and infection causative pathogens
2.1 Biofilm Formation
Biofilms are cluster of bacterial cells that are formed in 4 stages, as a defence mechanism
by the microorganisms, on the surface of the implant.
Figure 5: Illustration showing the stages of biofilm formation
2.1.1 Reversible Attachment
Within seconds of implanting the device, proteins, fibres, lipids and polysaccharides bind
to its surface and form a conditioning film [Gupta et al. (2015)]. When the planktonic
cells come in vicinity to this adhesive layer, they use physical forces, such as Van der
Waals interactions or use appendages to bind to the substratum.
2.1.2 Irreversible Attachment
The loosely packed cells become irreversible, when the attraction between the layers are
greater than the repulsive forces. This adhesion is mediated by protein such as SasX,
fibronectin, fibrinogen and laminin. During this phase, secondary messengers like di-c-
GMP, trigger the bacterial cells to secrete Extracellular Polysaccharide Matrix substances
4

6. (EPS), that engulfs the microbial colony, to enhance stability of the biofilm structure, to
provide nutrients for the cells and to protect the microbes from the external environment.
2.1.3 Maturation
Upon strong adhesion to the implant surface, the microbial cells will multiply in num-
bers, to form a thickness of 10m. Bacterial cells will communicate among each other, by
releasing autoinducer signals like N-acetylated homoserine lactone (AHL), resulting in mi-
crobial growth to a size of 100m. Next, quorum sensing (QS) measures this accumulated
signals, recognizes the high population density and develops microbial subpopulations
through phenotypic variations, which enhances the persistence of infections [Solano et al.
(2014)].
2.1.4 Dispersion
The released signals activate QS system that further induces serine protease activity to
mediate dispersal. Once dispersed, the bacterial cells scatter to other part of the surfaces
and start the process all over, thus worsening the effects of the microbial infections.
2.2 Consequences of Biofilm
Microbial films lead to tissue damage, spreading of pathogen to other sites, inflamma-
tory response and device malfunctioning. In orthopedic implants, microbial interventions
result in a serious condition of osteomyelitis, along with bone and tissue rupture [Veer-
achamy et al. (2014)]. During heart valve implantation, tissue damage occurs, that leads
to the settlement of fibrin and platelet at the surgical site and on the valve surface result-
ing in a condition called Endocarditis [Jung et al. (2012)]. This is complicated to treat,
because blood is not directly supplied to the valves, thus both white blood cells and drug
substances that rely on the blood system for reaching the infection site cannot remove
the biofilm. Urinary catheters are used to collect urine from long term patients, either in
a closed plastic bag or in an open vessel, thus increasing the vulnerability to infections.
In Europe, 97% of the deaths due to nosocomial infections are caused due to catheters.
Biofilm formation on these catheters cause urinary tract infections that leads to cystitis,
chronic renal infection, acute pyelonephritis, chronic prostatitis and death [Kirmusaoglu
et al. (2017)]. In breast implants, microbial infections would form capsular fibrosis, and
eventual contracture [E Steiert et al. (2013)].
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7. 2.3 Therapeutic Approaches
Figure 6: Schematic representation of strategies that are currently being investigated
Biofilms can be detected using blood tests, CT scan, MRIs or spectroscopy, but there are
very few symptoms at early stage infection, hence making it difficult to be diagnosed.
Few emerging diagnostic methods include using nanoparticles, and ultrasound. Conven-
tional treatment methods, like replacement, revision or antibiotic administration are less
effective, costly and require high dosage of antibiotics, because it is difficult for the drug
to penetrate through the EPS shield.
Moreover, the biofilm protects bacteria from the host immune response, phagocytosis,
UV radiation, pH stress, and chemical exposure, thus making it all inadequate. These
barriers has led to the development of novel strategies, currently in the clinical inves-
tigation stage, to accompany antibiotics, such as electromagnetic methods, bioacoustics
effect and photodynamic therapy, as outlined by Zohra Khatoon, et al., 2018 [Khatoon
et al. (2018)].
del Pozo et al. (2008) have demonstrated the effectiveness of electromagnetic methods,
in which electric fields are introduced on the bacterial cells, to breakdown the biofilm
through electroporation. The disintegrated cells are then destroyed by using antibiotic
or host immune response and this combined treatment is called as a bioelectric effect.
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8. Figure 7: Schematic diagram illustrating the detection of biofilm using surface acoustic
wave (SAW) and treatment based on the bioelectric effect
Another combinatorial method termed as Bioacoustics effect has been proven to re-
move 85% of biofilms associated to catheters, by enhancing the efficiency of antibiotics
through ultrasonication [Kopel et al. (2011)]. In this process, drug substances are en-
capsulated in a microbubble, injected into the bloodstream and upon reaching the site,
acoustic waves (300Hz) are used to transport the substances through the EPS layer, to
destroy the pathogens. Research by Chopra et al. (2017) has utilised acoustic wave in the
treatment of biofilm on knee implant, in which the target area is exposed to >100KHz
AMF, which instigates thermal destruction of the biofilm without damaging the tissue.
Figure 8: High frequency alternating magnetic fields (AMF) to treat biofilm
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9. Photodynamic therapy, as demonstrated by Poto et al. (2009), kills microbes by ex-
posing it to photosensitizer drug and visible light, which in combination produces radicals
and reactive oxygen species that are antimicrobial. However, further investigation is re-
quired for this method with regards to cytotoxicity.
Figure 9: Illustration of photodynamic treatment on dental implant biofilm
2.4 Novel Prevention Strategies
Futuristic methods are being developed to fill the unmet gap created by current and past
strategies to prevent adverse effects of biofilm.
Figure 10: Comparison between past, present and future prevention techniques
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10. 2.4.1 Implant surface materials
Implant surface is important in restricting infections and various nanofabrication tech-
niques such as deposition, etching and lithography are used to alter the surface properties
to limit microbial adhesion, on the basis of biocompatibility, roughness, hydrophilicity,
charge, energy and reactivity [Fox et al. (2019)].
Figure 11: Classes of materials used in implants and their advantages and disadvantages
2.4.2 Antibiofilm coatings
Antibiofilm coatings on the implant surface is a favourable prevention approach, under
which, one of the widely used method is antibiotic hydroxyapatite coating [Veerachamy
et al. (2014)]. Antimicrobial peptide (AMP) coatings are presently being investigated
a lot, because of their efficiency to disrupt resistant bacteria. Research by Atefyekta
et al. (2019) suggest that, AMP exhibits high bactericidal property, when grafted with
elastin-like polypeptide coating on the substratum. AMP are covalently immobilised on
the surface and through electrostatic forces and hydrophobic interactions, creates pores
on the cell membrane of the bacteria, promoting cell leakage and eventual lysis. Re-
search conducted by Xie et al. (2019) depicted successful reduction of microbes, when
silver nanoparticles were coated on the implant. The Ag+ particles tend to penetrate
into the bacteria, react with DNA, rupture the respiratory system, eventually causing cell
death. Research by Kim et al. (2008) suggest that combining tobramycin/ciprofloxacin
with silver will increase microbial killing efficacy by more than 200%. Recently, an ex-
periment conducted by Sadrearhami et al. (2019), observed that, coating substrate with
polydopamine and polyethylene glycol, produces nitric oxide that prevented bacterial
adhesion by 96%.
2.4.3 Quorum sensing inhibition
QS plays a major role in bacterial growth, and hence, by restricting the synthesis of au-
toinducers, degrading the signalling molecules or by replacing the signalling molecule with
an antagonist, that competes for the binding site, cell communication can be restricted,
thus inhibiting biofilm formation [Brackman & Coenye (2014)].
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11. Figure 12: Prevention of biofilm formation through Quorum quenching
Some of the inhibitory agents are furanones, glycosylation reagents of flavonoids, and
signal degrading enzymes such as AHL acylase, lactonase, and oxidoreductases [Izano
et al. (2008)]. Surface adhesion and extracellular matrix layer formation are mediated
by the function of poly-(1,6)-N-acetyl-D-glucosamine (PNAG) and extracellular DNA
(ecDNA). These two polymers can be defunctionalized by treating with dispersin B and
DNase I, which would result in EPS inhibition, and would sensitize the bacteria to the
killing by cetylpyridinium chloride, a cationic detergent [Raafat et al. (2019)].
2.4.4 Vaccines
Figure 13: Illustration of biofilm disruption by using monoclonal antibodies
Until recently, vaccination has not been successful in the case of S. aureus, because of its
ability to adapt and express phenotypic variation. However, an experiment conducted
on rabbits by Brady et al. (2011), has depicted 87.5% eradication of these biofilms, by
using antibiotics combined with a quadrivalent vaccine targeting the glucosaminidase, a
conserved protein and a transporter lipoprotein. Also, an experiment conducted on mice
10

12. by Ortines et al. (2018), to neutralise the alpha-toxin, a key virulence factor in S. aureus,
by using anti-AT Mab, has emerged to be a success.
3 Discussion
Unlike the existing approaches to eradicate or prevent biofilm, the current methods dis-
cussed are more efficient, non-invasive, and requires lower antibiotic dosages, thus ad-
dressing the issue of antibiotic resistant microbes. Upcoming prevention strategies con-
centrates on using the right device material, enhancing the host immune response and
utilising natural products for biofilm prevention. Vaccination and anti-biofilm coatings
are some of the most promising prevention approaches, but as it is in investigational
stages, it needs to pass the hurdle of translating from animal models to human studies.
4 Conclusion
Biofilms are a major health hazard and an economic burden, because of its physiochemi-
cal properties, that makes it difficult to treat and prevent. Irrespective of the risk class of
the device and the indwelling site, implants are highly crucial, thus emphasizing on the
need to maintain it properly. One cannot wait until the bacteria develops into biofilms,
since treatment would pose a major threat to the patient health, will be expensive, and
recurrence of infection will also be high. Also, a huge portion of these prevention and
treatment strategies require the administration of antibiotics, which increases the risk of
development of resistant microbes, that causes 33000 deaths annually in Europe. From
this essay, we can understand that, these adverse effects, can be avoided, by modifying the
implant surface by using nanofabrication techniques, by applying appropriate antibiofilm
coating, and by developing novel vaccination techniques. Along with these factors, the
biomaterial chosen to make the implant should be highly biocompatible, smooth and hy-
drophilic, to enhance tissue integration, functionality of implant and to prevent microbial
infections.
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