on Staphylococcus aureus and Pseudomonas

ORIGINAL ARTICLE
Laser irradiation effect
on Staphylococcus aureus
and Pseudomonas
aeruginosa biofilms isolated
from venous leg ulcer
Marina Baffoni, Lucinda J Bessa, Rossella Grande, Mara Di Giulio,
Matteo Mongelli, Antonio Ciarelli, Luigina Cellini
Baffoni M, Bessa LJ, Grande R, Di Giulio M, Mongelli M, Ciarelli A, Cellini L. Laser irradiation effect on
Staphylococcus aureus and Pseudomonas aeruginosa biofilms isolated from venous leg ulcer. Int Wound J 2011;
doi: 10.1111/j.1742-481X.2011.00910.x
ABSTRACT
Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, represent a significant cause
of morbidity in developed countries, predominantly in older patients. The aetiology of these wounds is probably
multifactorial, but the role of bacteria in their pathogenesis is still unclear. Moreover, the presence of bacterial
biofilms has been considered an important factor responsible for wounds chronicity. We aimed to investigate the
laser action as a possible biofilm eradicating strategy, in order to attempt an additional treatment to antibiotic
therapy to improve wound healing. In this work, the effect of near-infrared (NIR) laser was evaluated on mono
and polymicrobial biofilms produced by two pathogenic bacterial strains, Staphylococcus aureus PECHA10 and
Pseudomonas aeruginosa PECHA9, both isolated from a chronic venous leg ulcer. Laser effect was assessed by
biomass measurement, colony forming unit count and cell viability assay. It was shown that the laser treatment has
not affected the biofilms biomass neither the cell viability, although a small disruptive action was observed in the
structure of all biofilms tested. A reduction on cell growth was observed in S. aureus and in polymicrobial biofilms.
This work represents an initial in vitro approach to study the influence of NIR laser treatment on bacterial biofilms
in order to explain its potentially advantageous effects in the healing process of chronic infected wounds.
Key words: Biofilm communities • Chronic wound • Laser irradiation • Pseudomonas aeruginosa • Staphylococcus aureus
Authors: M Baffoni, BS, Department of Biomedical Sciences, University ‘G. d’Annunzio’, Chieti-Pescara, Chieti, Italy; LJ Bessa, BS,
Department of Biomedical Sciences, University ‘G. d’Annunzio’, Chieti-Pescara, Chieti, Italy; R Grande, PhD, Department of Drug Sciences,
University ‘G. d’Annunzio’, Chieti-Pescara, Chieti, Italy; M Di Giulio, PhD, Department of Drug Sciences, University ‘G. d’Annunzio’,
Chieti-Pescara, Chieti, Italy; L Cellini, PhD, Department of Drug Sciences, University ‘G. d’Annunzio’, Chieti-Pescara, Chieti, Italy; M
Mongelli, MD, Division of Vascular Surgery, ‘‘Santo Spirito’’ Hospital of Pescara, Italy; A Ciarelli, MD, Division of Vascular Surgery, ‘‘Santo
Spirito’’ Hospital of Pescara, Italy
Address for correspondence: L Cellini, Department of Drug Sciences, School of Pharmacy, University ‘G. d’Annunzio’, Chieti-Pescara,
Via dei Vestini 31, Chieti, Italy
E-mail: [email protected]
Marina Baffoni and Lucinda J. Bessa contributed equally to this work.
© 2011 The Authors
© 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc
• International Wound Journal • doi: 10.1111/j.1742-481X.2011.00910.x
1
Laser effect on biofilms isolated from venous leg ulcer
INTRODUCTION
Key Points
• biofilm presence in wounds
have been considered an important factor responsible for
wounds chronicity
• antibacterial therapies are often
unable to improve the chronic
wounds outcome
• laser irradiation used in wound
debridement could promote the
biofilm eradication and contribute to the wound-healing
process
• the effect of near-infrared
(NIR) laser was evaluated in
vitro on mono and polymicrobial biofilms produced by
two pathogenic bacteria strains
isolated both from a chronic
venous leg ulcer
• laser effect was evaluated by
biomass measurement, colony
forming unit count and cell
viability assay
2
Chronic wounds, including diabetic foot ulcers,
pressure ulcers and venous leg ulcers affect
1% to 2% of the population in developed
countries and represent a significant cause
of morbidity predominantly in patients older
than 60 years (1–3).
The aetiology of these wounds is yet poorly
understood; however, it is probably multifactorial. It has been shown that inflammatory and infectious processes contribute to the
chronic state. Normally, the infection results
from an increased host susceptibility or from
an increased microbial concentration and virulence of the strain, although both factors might
be present (4).
The role played by microorganisms in
chronic non healing wounds is still not
entirely known (5). The microbial bioburden,
the microbial species and the biofilm formation
within the wound are important factors that
can delay the healing process. Although it is
widely accepted and well documented that
both acute and chronic wounds are more
susceptible to infection when the bioburden
within the wound bed reaches a ‘critical
level’ (6–8), recent studies have shown that
bacteria are randomly distributed within the
wound and consequently the assessment of
the true microbial density in a wound is often
biased (3,4).
It is known that the microflora of chronic
wounds comprises multiple species. Different bacterial profiling studies using standard culturing (9–12) and modern molecular
microbiological methods (13–16) have documented that predominant microorganisms in
chronic wounds include Staphylococcus spp.,
Pseudomonas spp., Streptococcus spp., Enterococcus spp., Corynebacterium spp. and various
anaerobes and suggest that the specificity of
microorganisms is more important than bacterial bioburden to delay or even prevent the
healing process.
Most of the chronic wound pathogens, such
as Staphylococcus aureus and Pseudomonas aeruginosa, are typical biofilm producers (17,18). The
presence of P. aeruginosa biofilm in wounds has
been recently documented and considered as
an important factor responsible for wounds
chronicity (19–21). Bacteria in biofilm communities show significantly greater resistance to
traditional antimicrobial therapies than their
planktonic counterparts, therefore removing
biofilm may be important in the management
of chronic wounds and new biofilm eradication strategies to treat chronic wounds are
demanded (22–25).
Currently, laser therapy to improve wound
healing opens a new perspective in this
research area (26). Laser irradiation can
enhance the release of growth factors from
fibroblasts and stimulate the wounded cells to
recover their functions and increase their in
vitro proliferation (27,28).
Although some studies have focused on
selective photodamage induced in bacterial
and fungal cells (29–31) by near-infrared (NIR)
irradiation, to date, little is known about the
effect of laser irradiation on bacterial biofilms
present in infected wounds.
In this work, a possible killing or disruptive
effect of NIR laser was evaluated on mono
and polymicrobial biofilms, formed by two
pathogenic bacteria strains both isolated from
a chronic venous leg ulcer.
As antibiotic therapy resistance constitutes
nowadays, more than never, a problem to overcome, alternative bacterial eradication treatments are demanded. This work represents
an initial attempt to study new mechanisms
that might be involved in the modification of
bacterial biofilms structure.
MATERIALS AND METHODS
Bacterial strains
Two bacterial strains, Staphylococcus aureus
PECHA10 and Pseudomonas aeruginosa PECHA9,
were isolated from the wound of a patient with
chronic venous leg ulcer and used for the in
vitro biofilm formation. Swab sample was cultured on blood agar, McConkey agar, Mannitol
Salt agar and Sabouraud agar (Oxoid, Milan,
Italy) for bacteria and yeast recovery. The two
isolated strains were identified by VITEK 2
Technology (Biomerieux, Florence, Italy) and
stored in Microbank™ system (Biolife, Milan,
Italy) at −80◦ C.
Biofilm growth condition
The two clinical strains used for the experiments were separately harvested in Tryptic
Soy Broth (TSB; Oxoid, Milan, Italy) and incubated overnight aerobically at 37◦ C. In order to
obtain log-phase broth cultures, the overnight
cultures were diluted 1:5 in fresh TSB supplemented with 0·5% (w/v) glucose (Sigma
© 2011 The Authors
© 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc
Laser effect on biofilms isolated from venous leg ulcer
Aldrich, Milan, Italy) and incubated aerobically
for 2 hours at 37◦ C under shaking (100 rpm);
these cultures were then adjusted to an optical
density at 600 nm (OD600 ) of 0·1 corresponding
to approximately 108 CFU/ml; a final dilution of 1:100 in TSB supplemented with 0·5%
(w/v) glucose was performed for each strain.
Such standardised broth cultures were used to
prepare a mixture 1:1 (v/v) of the two strains.
The above prepared cultures of S. aureus
PECHA10, P. aeruginosa PECHA9 and their
mixture were then inoculated into a collagen type I-coated, flat-bottomed 96-well
polystyrene microtiter plate (Iwaki, Tokyo,
Japan) (200 μl/well) for biofilm formation
assay and colony forming units (CFU) count.
Moreover, 2 ml was poured into collagen
type I-coated 35 mm of diameter polystyrene
plates (Iwaki, Tokyo, Japan) for cell viability
assay. The microplates and plates were incubated aerobically for 24 hours at 37◦ C.
Laser treatment
Twenty-four hours preformed biofilms, grown
on TSB supplemented with 0·5% (w/v) glucose, were submitted to a laser treatment
with an NIR diode laser (A.R.C. Laser GmbH,
Nürnberg, Germany), with a wavelength of
980 nm coupled to a 400 nm optical fibre. The
NIR laser was applied with an energy level of
10 W, with a distance of 5 cm, delivering a total
energy density of 148 J/cm2 . These parameters were selected in accordance to the clinical
experimental practice performed in the Division of Vascular Surgery of ‘‘Santo Spirito’’
Hospital of Pescara to treat infected chronic
wounds.
After the laser treatment, the biofilms were
investigated for biomass measurement, CFU
count and cell viability assay in respect to the
untreated corresponding controls.
All tests were carried out for two independent experiments, and each experiment was
performed in triplicate.
Biofilm formation assay
The treated biofilms and respective controls
were analysed for the biomass measurement.
Planktonic cells were removed and biofilms
were rinsed with 300 μl of phosphate buffered
saline (PBS), pH 7·3, fixed by air drying and
stained with 200 μl of crystal violet 0·5% (w/v)
for 2 minutes. The stained biofilms were rinsed
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© 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc
with 300 μl of PBS, air dried, eluted with 200 μl
of acetic acid 33% and then the optical density
was measured at 595 nm using a microplate
reader (SAFAS, Munich, Germany). The results
were expressed as average of OD595 values of
three replicates (32).
The statistical significance of difference
between controls and experimental groups was
evaluated using Student’s t test. Probability
levels of <0·05 were considered statistically
significant.
Cell viability assay
For the evaluation of cells viability, both
planktonic and sessile phases of S. aureus
PECHA10, P. aeruginosa PECHA9 and of their
polymicrobial biofilms were stained using
the Live/Dead BactLight bacterial viability
kit (Invitrogen, Milan, Italy). The planktonic
cells were removed from each plate, centrifuged at 8000 rpm for 10 minutes at 4◦ C
and resuspended in 500 μl of the appropriate mixture of SYTO 9 and propidium iodide
stains, followed by 20 minutes of incubation
at room temperature in the dark, as indicated by the manufacturer’s instructions. Five
microlitres of the stained suspensions were
immediately examined using a fluorescent
Leica 4000 DM microscope (Leica Microsystems, Wetzlar, Germany). Instead, the sessile
cells were washed with 1 ml of PBS and stained
with 500 μl of the appropriate mixture of SYTO
9 and propidium iodide stains, incubated for 20
minutes at room temperature in the dark; then,
were rinsed with 1 ml of PBS and examined
under fluorescence. Images were recorded at
an emission wavelength of 500 nm for SYTO 9
(green fluorescence) and of 635 nm for propidium iodide (red fluorescence).
Microscopic observations were carried out
in triplicate. Ten fields of view randomly
chosen for each slide were examined. The
counts were repeated independently by three
microbiologists by using an image analysis
software (QWIN, Leica Microsystems, Wetzlar,
Germany).
Colony forming unit count
The planktonic cells were removed from each
well of microplates, vortexed, serially diluted
(1:10) on TSB, plated on Tryptic Soy Agar (TSA)
(Oxoid, Milan, Italy) and incubated aerobically
for 24 hours at 37◦ C; the biofilms were rinsed
3
Laser effect on biofilms isolated from venous leg ulcer
with PBS; then, 100 μl of PBS was added to
each well to collect cells using a cell scraper
(Corning, Turin, Italy). Each sample was then
vortexed, serially diluted (1:10) on TSB, plated
on TSA and incubated aerobically for 24 hours
at 37◦ C.
The concentration was calculated as mean
value of five replicates for both mono
and polymicrobial biofilms and reported as
CFU/ml. The statistical significance of differences between controls and experimental
groups was evaluated using Student’s t test.
Probability levels of <0·05 were considered
statistically significant.
RESULTS
In this study, the effect of NIR laser
on mono and polymicrobial biofilms of S.
aureus PECHA10 and P. aeruginosa PECHA9
pathogenic bacteria, isolated from a chronic
venous leg ulcer, was evaluated.
The laser effect on 24-hour mature biofilms,
grown under static conditions, showed no
significant differences in terms of biomass
reduction in both mono and polymicrobial
biofilms when compared to the controls
(Figure 1).
Similarly, the cell viability of S. aureus
PECHA10 and P. aeruginosa PECHA9 in mono
and polymicrobial biofilms was not affected by
laser irradiation as shown in Figure 2 in which
a large prevalence of live cells was detected.
The QWIN analysis software (Leica Microsystems, Wetzlar, Germany) recorded a similar
percentage of live cells (from 98 to 99%) in sessile and planktonic phases of both treated and
untreated mono and polymicrobial biofilms.
A qualitative evaluation of Live/Dead images
displayed a modification on the compactness
of treated biofilms in respect to the controls,
in particular in P. aeruginosa biofilm and in
polymicrobial biofilm as shown in Figure 2C,
D and E, F, respectively. On the contrary, a
reduction on bacterial growth of treated sessile and planktonic cells was observed in some
cases (Table 1). In particular, the CFU count of
sessile and planktonic S. aureus PECHA10 was
significantly lower (P < 0·05) in treated samples than in respective controls. A similar trend
was also noticed in the polymicrobial biofilm
(P = 0·16 and 0·01 for sessile and planktonic
phases, respectively), whereas no reduction of
bacterial growth was recorded in P. aeruginosa
PECHA9 biofilm (P = 0·42 and 0·09 for sessile
and planktonic phases, respectively).
DISCUSSION
Presence of infection within the wound bed
can greatly contribute to a state of chronicity
Figure 1. Laser effect on Staphylococcus aureus PECHA10, Pseudomonas aeruginosa PECHA9 and their mixture biofilms measured
spectrophotometrically by OD595 (mean ± SD) causing no significant reduction in biofilm (P < 0·05).
4
© 2011 The Authors
© 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc
Laser effect on biofilms isolated from venous leg ulcer
Figure 2. Representative fluorescence micrographs of Staphylococcus aureus PECHA10, Pseudomonas aeruginosa PECHA9 and
their mixture biofilms after 24 hours of incubation, showing the preservation of cells viability with and without laser treatment. Laser
non treated samples (on the left): Biofilm (A) and planktonic cells (a) of S. aureus PECHA10; biofilm (C) and planktonic cells (c) of
P . aeruginosa PECHA9 and biofilm (E) and planktonic cells (e) of mixed species. Laser treated samples (on the right): Biofilm (B) and
planktonic cells (b) of S. aureus PECHA10; biofilm (D) and planktonic cells (d) of P. aeruginosa PECHA9 and biofilm (F) and planktonic
cells (f) of mixed species. Scale bars = 5 μm.
Table 1 Colony forming unit count (CFU/ml) of Staphylococcus aureus PECHA10, Pseudomonas aeruginosa PECHA9 and their
mixture detected in the sessile and planktonic phases
Control
Sessile
Planktonic
Sessile
Planktonic
2·1 × 109
5·2 × 109
1.0 × 1010
1·2 × 108
1·2 × 1010
2·2 × 1010
1·2 × 108
6·6 × 109
3·2 × 109
6.0 × 107
3·3 × 109
3·1 × 109
Species
S. aureus PECHA10
P. aeruginosa PECHA9
S. aureus PECHA10/P. aeruginosa PECHA9
Laser treated
and also delay the wound-healing process (4).
Currently, wound debridement, along with
dressings containing antimicrobial agents and
systemic antimicrobial therapy represent the
main available means used by clinicians in
© 2011 The Authors
© 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc
the wound infection handling; however, these
treatment strategies usually appear to be
ineffective in the infection eradication (7).
Many studies (4,15) have pointed out
biofilms as a major factor for the chronic
5
Laser effect on biofilms isolated from venous leg ulcer
behaviour of wounds. Therefore, the presence,
within a wound, of microorganisms capable
to form biofilms is definitely an important
contributing factor to the difficulty of eradication. The isolates used in our assays,
P. aeruginosa and S. aureus, both showed to
be good biofilm producers, contributed to the
chronicity state of the patient’s leg ulcer. Moreover, the occurrence of polymicrobial biofilms
may be an additional problem, hampering even
more the eradication and therefore the healing (21). Thus, to verify the laser effect, we
performed not only monomicrobial biofilms,
but also polymicrobial ones.
The laser treatment applied to 24 hour
mature monomicrobial and polymicrobial
biofilms did not affect the biofilm cells viability and biomass, although a small reduction on
the compactness of those biofilms was recorded
at microscopic observation. These results can
be explained by the NIR laser inability to
pass through the exopolymeric substances of
mono and polymicrobial biofilms, and thus non
affecting cell viability. In any case, the modifications recorded in the compactness of treated
biofilms in respect to the corresponding non
treated controls might be associated to a mild
disruptive effect of NIR laser on the top of the
biofilm. At this point, it will be interesting to
use antimicrobial agents after the laser treatment, which could facilitate their access to the
bacterial target.
In accordance to our results, non effective
killing action in methicillin-resistant S. aureus
biofilm was shown by Krespi et al. (33) by
using NIR laser alone; however, this effect was
achieved when a pretreatment with a shock
wave laser was performed.
A reduction on CFU count was significant
only in some cases analysed as in both
treated phases of S. aureus PECHA10 and
in treated planktonic phase of polymicrobial
biofilm in respect to controls. This result can be
interpreted by the presence of a viable but non
culturable cell subpopulation – a reversible,
quiescent state typically associated to biofilms
of numerous bacteria, which exhibit a loss of
culturability. In this ‘dormant state’, bacteria
are characterised by the presence of viable cells,
observed as green fluorescence at microscope
examination, unable to replicate and therefore
unable to be cultivated (34,35).
Although the laser benefits are mostly due
to its ability to promote regeneration of
6
damaged cells, laser irradiation may modify
the metabolic features in the microbial cells
and therefore might constitute a stressful
condition inducing a small part of the biofilm
population to enter a ‘persister’ cell state (36).
This particular state could lead to a restraint
in bacterial cell proliferation, which may
contribute to a minor recruitment of immune
cells in the bed wound. Therefore, wound
healing could be favoured through a slowdown
in the inflammatory process together with the
tissue regeneration effect of laser.
Our in vitro experimental model was performed using collagen type I-coated plates
in order to better resemble the in vivo bacterial growth conditions. Similarly, Werthén
et al. (37) have also developed a new in vitro
model to investigate bacterial biofilms in
chronic wounds, with bacteria growing as
biofilm aggregates in a collagen gel matrix.
Although, our results do not point out a
considerable effect of NIR laser on S. aureus,
P. aeruginosa and their polymicrobial biofilms,
they may highlight the need of performing
new combined strategies to improve the
healing of infected wounds instead of using
pharmacological or laser therapies alone.
Further work should consist in performing
assays that can better mimic the wound bed
and that also combine the use of different types
of laser together with antimicrobial therapy.
ACKNOWLEDGEMENTS
This study was supported by a grant
awarded by Ministero Istruzione, Università
e Ricerca, Italy.
The authors thank Soraya Di Bartolomeo,
Emanuela Di Campli and Alessandra Felici for
their technical support.
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