research

Propagation of anaerobic bacteria

within an aerobic multi-species
chronic wound biolm model
Objective: Most chronic wound biofilms have been shown to have significant populations of
anaerobes. In order to better screen antimicrobial and antibiofilm therapeutics, we evaluated the ability
of key anaerobes to incorporate and propagate within our aerobic chronic wound biofilm.
Method: We had previously developed a rapid model to simulate polymicrobial chronic wound

biofilms. This model incorporated meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant

Enterococcus faecalis (VRE) and Pseudomonas aeruginosa. The model was used along with a variety of
anaerobes to determine whether this biofilm model would support propagation of anaerobes similar to
that we have identified in chronic wounds.
Results: Using our previously defined Lubbock Chronic Wound Biofilm (LCWB) model combined

with quantitative PCR, anaerobic bacteria were shown to proliferate through integration into the biofilm
under aerobic conditions. Using electron microscopy we show close association between aerobes and
anaerobes within the biofilm suggesting a synergistic relationship.
Conclusion: We have expanded the utility of the LCBW to show the ability of clinically significant

anaerobic bacteria to thrive in aerobic conditions. The expansion of this model can further simulate the
functional characteristics of chronic wound pathogenic biofilms and the species that dwell within them
allowing improved ability to evaluate therapeutics that target anaerobes.
Declaration of interest: None.

chronic wounds; biofilms; anaerobes; MRSA; VRE; Pseudomonas aeruginosa

C
Y. Sun, PhD, Molecular hronic wounds affect millions of peo-
Microbiologist, Research ple, incur annual health care costs in
and Testing Laboratory,
Lubbock, Texas, US; the billions of dollars and kill hundreds
E. Smith, BSc, Laboratory of thousands of patients annually in
Technician, Research and the US.1-3 It has recently been proposed
Testing Laboratory, that the presence of multi-species pathogenic bio-
Lubbock,TX, US;
films within these wounds contributes to their
R. Wolcott, MD, Medical
Director, Southwest chronicity.
Regional Wound Care Chronic wound pathogenic biofilms are highly
Center, Lubbock, TX, US; resistant and adaptable systems. Contributing to
S.E. Dowd, PhD, Director, this adaptability is the functional diversity associ-
Medical Biofilm Research
Institute and Research and ated with polymicrobial biofilms.4 The genetic
Testing Laboratory, diversity of this polymicrobial biofilm community
Lubbock, TX, US. potentially enhances its pathogenic potential. The
Email: sdowd@ effects include:
pathogenresearch.org Enhanced bacterial resistance to antibiotics for
example, some bacteria may have resistance factors,
which they release into the biofilm. In this way,
they confer this resistance to neighbouring bacterial
populations without resistance
Increased numbers of available virulence factors
Bacteria thriving in environments that would oth-
erwise prevent their propagation for example,
aerobic bacteria can use up available oxygen, allow-
ing anaerobic bacteria to propagate
Participation in localised nutrient cycling for
example, some bacteria can release key nutrients
that can be used by neighbouring populations, or
some populations can use and degrade compounds
that might be harmful to other populations.
Ultimately, polymicrobial biofilms are a commu-
nity made up of many different genera and species
of bacteria that work together to increase the adapt-
ability and even the pathogenicity of the entire bio-
film community.5,6 This suggests that the hosts abil-
ity to control these multi-species entities may
decrease in accordance with the functional diversity
of the biofilm community.2,4,7,8
Three important aerobic and facultative species
associated with multi-species chronic wound bio-
films are Pseudomonas aeruginosa, Enterococcus faec-
alis and Staphylococcus aureus.9 Also of great signifi-
cance in this microbial community, and perhaps
more intriguing, are anaerobic bacteria. In some
wound types, such as diabetic foot ulcers (DFUs),
venous leg ulcers (VLUs) and surgical site infec-
tions (SSIs), anaerobes are often the predominant
species and may contribute greatly to the biofilm
pathogenicity.2,4,7,10
An anaerobic organism does not propagate as a
pure culture in the presence of oxygen. However,
the underlying reason for their classification as
anaerobes is that they do not use oxygen as a termi-
nal electron acceptor.

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Some anaerobes can be killed by the presence of
oxygen.11,12 Consequently, their survival in the aero-
bic environment of oral biofilms6 and sludge flocs in
waste water treatment reactors5 is cause for thought.
These occurrences have been attributed to a pos-
sible oxygen gradient within the biofilm. This gra-
dient is created by the primary aerotolerant species
within the biofilm, and allows the anaerobes to
integrate and propagate there. As the biofilm devel-
ops and grows in thickness, the outer layers are
exposed to more oxygen. However, even a few
microns deep into the biofilm the diffusion of oxy-
gen is hindered by the density of the biofilm
matrix, while anaerobic bacterial populations con-
sume the oxygen that does diffuse into it. This oxy-
gen gradient therefore creates an environment in
which strict anaerobes can survive and propagate,
contributing to the genetic diversity and thus fit-
ness of the pathogenic community.
Furthermore, the ability of bacteria to communi-
cate with each other within a biofilm supports the
theory that anaerobic bacteria can thrive within
global aerobic conditions by cross-feeding and
enzyme complementation until an anaerobic envi-
ronment has been established.6
This study provides the first example of this phe-
nomenon within an in-vitro aerobic wound biofilm
model the Lubbock chronic wound biofilm,
LCWB. (We expanded the LCWB model to include
anaerobic bacteria.)
As shown previously, this model can be used to
test multiple antimicrobial substances that might
have important clinical implications. Confirmation
of the similarity of the LCWB model to in vivo
chronic wound pathogenic biofilms9 further sup-
ports the validity of first-line evaluation of antimi-
crobial substances on polymicrobial biofilm com-
munities that include key anaerobic bacteria.
Materials and method
Bacteria
We selected several anaerobic bacteria as type spe-
cies for chronic wounds: Peptoniphilus ivorii, Pepto-
streptococcus anaerobius, Anaerococcus lactolyticus,
Finegoldia magna and Clostridium perfringens.
Peptoniphilus ivorii, Peptostreptococcus anaerobius,
Anaerococcus lactolyticus and Finegoldia magna are
Gram-positive anaerobic cocci,13 which can cause
serious opportunistic infections. Indeed, they are
collected from clinical samples more often than any
other anaerobic bacteria, except Bacteroides,14 and
are the chief bacterial species in certain chronic
wound types.4,7
For example, Peptoniphilus ivorii and Finegoldia
magna were shown to be major bacterial contributors
to biofilm enhanced infection and subsequent con-
tributors to the chronicity of DFUs, SSIs and PUs.4,7,15,16
Finegoldia magna has been found in the genitourinary
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research
tract of renal transplant recipients17 and Peptostrepto-
coccus anaerobius has been shown to cause brain, liv-
er, breast and lung abscesses.18-20 Clostridium perfrin-
gens is a Gram-positive anaerobic rod, which has
been found in many wound types including gas gan-
grene.21 In addition, these bacteria have all been
identified in chronic wound biofilms.4,7,15,16,22
Pseudomonas aeruginosa PAO1 (ATCC number:
BAA-47), Enterococcus faecalis V583 (700802) and
Staphylococcus aureus Mu50 (700699) were main-
tained in initial cryostock cultures, resuscitated on
tryptic soy agar (TSA, Sigma Chemical, St Louis,
MO, US) plates combined with antibiotics to target
resistance in each bacterial species. Ampicillin
(250g/ml) was used for P. aeruginosa, vancomycin
(40g/ml) for Enterococcus faecalis and Difco Tellur-
ite Glycine agar (BD & Co., Sparks, MD, US) for Sta-
phylococcus aureus.
Isolated colonies were inoculated in TSB and
grown overnight at 37C with shaking at 140 revo-
lutions per minute (rpm).
All anaerobes were chosen as representatives
because they had previously been associated with
chronic wounds.4,7,15,16 Peptoniphilus ivorii (BAA-
602), Peptostreptococcus anaerobius strain VPI 4330
(27337), Anaerococcus lactolyticus (51172) and Fine-
goldia magna (29328) were resuscitated on 5% RCM
blood agar plates under a miniMACS anaerobic
workstation (Don Whitley Scientific, MD, US) with
bioblend anaerobic biological incubation atmos-
phere (10% carbon dioxide, 10% hydrogen, 80%
nitrogen; Airgas, PA, US) at 37C. Clostridium perf-
ringens strain S (13124) was resuscitated on rein-
forced clostridial agar (RCA, Sigma Chemical) and
isolated colonies were grown overnight in RCM
broth (RCM, Oxoid, Hampshire, England) in the
anaerobic chamber. Growth medium chosen was
based on literature and facilitation of growth after
13 days incubation.
Biofilm formation media
The LCWB9 was utilised for all tests. Bolton broth
(Oxoid, Hampshire, England) with 48% heparinised
bovine plasma and 2% laked blood (HemoStat Labo-
ratories, Dixon, CA, US) was used as the biofilm for-
mation media.
7ml biofilm formation media was aseptically dis-
pensed in autoclaved test tubes. Overnight cultures
of the bacteria were diluted 100X in TSA and OD600
measured using a GENESYS-20 spectrophotometer
(Thermo Scientific, MA, US). (A spectrophotometer
uses wavelengths to measure optical density [OD] of
suspension cultures. Here, a 600nm wavelength is
used to indicate cell mass/number, after construc-
tion and calibration of a standard curve.)
Plate counts were performed using a Whitley
automatic spiral plater (Don Whitley Scientific, MD,
US). Bacterial counts were also performed using a
427
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modified protocol with the BacLight Viability Kits
(Invitrogen, OR, US) for fluorescence microscopy on
an Olympus BX51 microscope mounted with an
Olympus DP71 digital camera (Olympus Imaging
America., Center Valley, PA, US).
Bacterial-count normalised cultures of the bacte-
ria were mixed together (~1 x 105 CFU/ml average
concentration) and 10l of this mixture were inocu-
lated into glass test tubes. This was done by ejecting
the pipette tip into the tubes, which acted as a sur-
face for biofilm formation. The biofilms were then
grown at 37C in a shaker for 24 hours at 140rpm.
Bacteria were tested for growth, both at 24 and 48
hours. It was thought that the biofilm formation
would occur in 24 hours, and that by 48 hours there
would be a sufficiently anaerobic environment in
which anaerobes can grow and mature.
Electron microscopy
Scanning electron microscopy (SEM) of biofilm sam-
ples was performed as follows. Samples were fixed
(3% paraformaldehyde, 1.5% glutaraldehyde) for
one hour at room temperature. After dehydration in
an ethanol series, they were transferred to hexame-
thyldisilazane (HMDS) for drying. Dried samples
were mounted on aluminium stubs using alumini-
um paint, and sputter coated with gold. The speci-
mens were viewed and photographed with a Hitachi
S-3400N (Hitachi, Japan) SEM.
SEM was performed on biofilms containing Ente-
rococcus faecalis and Staphylococcus aureus (coccus)
and Clostridium perfringens (rod) to visualise mor-
phological differences.
DNA extraction
Biofilm samples were placed on dry ice until frozen
and then ground to a homogenous liquid using ster-
ile 15ml closed tissue grinder systems (FisherScien-
tific, TX, US) connected to a power drill for full
homogenisation. 500l Tris-EDTA buffer was added
to the tube and vortexed to wash biofilm off the
grinder pestle.
Samples were transferred to a 1.5ml Eppendorf
tube and 500l 0.1mm glass beads (Scientific Indus-
tries, NY, USA) were added for complete bacterial
lyses in a Qiagen TissueLyser (QIAGEN, CA, US), run
at 30Hz for five minutes. Samples were centrifuged
briefly and 350l RLT (with -ME) and 250l 100%
ethanol were added to a 100l aliquot to prepare
samples for DNA extraction.
This solution was added to a DNA spin column
and DNA recovery protocols were followed as
instructed in the QIAamp DNA Mini Kit (QIAGEN,
CA, USA) starting at step 7 of the tissue protocol.
DNA was eluted from the column with 100l water
and samples were diluted accordingly to a final con-
centration of 20ng/l for use with SYBR green-based
quantitative PCR (qPCR). SYBR green is a dye that
428
Journal of Wound Care. Downloaded from magonlinelibrary.com by 131.172.036.029 on June 15, 2016. For personal use only. No other uses without permission. . All rights reserved.
intercalates with double-stranded DNA. This inter-
calation causes the SYBR to fluoresce, allowing
quantification of DNA based on the relative amount
of fluorescence detected. qPCR is a molecular meth-
od that uses to primers to detect and quantify spe-
cific targets in the genomes of bacterial pathogens.
Primer design
Primer kits for qPCR for all organisms used in this
study were obtained from Research and Testing
Laboratory (Lubbock, TX, US). The primers
designed for detection of the anaerobic bacteria
were tested to ensure they did not give false posi-
tive results or cross react with any other bacteria
(aerobic or anaerobic) used in this study. The prim-
ers were also tested to ensure they had similar
detection sensitivities. Primers with the aerobic
primers, endpoint PCR assays and real-time PCR
linear assays were performed.
Genomic DNA was extracted from planktonic
cells using a QIAamp DNA mini kit (QIAGEN, CA,
US) with a modified protocol. DNA samples were
quantified using a Nanodrop spectrophotometer
(Nyxor Biotech, Paris, France).
For cross-reactivity/specificity tests, genomic DNA
from each of the bacterial strains was diluted to
20ng/l and tested against each primer set.
For efficiency tests, genomic DNA was diluted to
concentrations ranging from 0.25 to 50ng/l per
reaction and each species of DNA was individually
tested for linearity.
Primers were chosen that were both specific and
which had similar amplification efficiencies to allow
accurate comparisons.
Quantitative PCR
The relative ratios of each bacterium were evaluated
using the ABI 7500 real-time PCR system (Applied
Biosystems, CA, USA). Primers for each bacterial
species, along with Bio-Rad iTaq SYBR-Green Super-
mix with ROX, were used for 25l real-time qPCR
reactions as follows: 95C for 10 minutes, and 40
cycles of 95C for 15 seconds and 60C for 60 sec-
onds. All reactions were performed in triplicate. The
relative genome copy number ratios were calculated
and analysed (User Bulletin #2, ABI PRISM 7700
Sequence Detection System).
In brief, the threshold cycle (Ct value) of the tar-
get genes in different samples was obtained after
qPCR reaction. The Ct value of the calibrator (the
sample with the highest Ct value) was subtracted
from all the other samples to produce the ddCt val-
ue. Two to the -ddCt power (2-ddCt) was taken for
every sample and used to evaluate relative ratios of
each bacteria.
Ct is the intersection between an amplification
curve and a threshold line. It is a relative measure
of the concentration of target (in this case, specific
J O U R N A L O F WO U N D C A R E V O L 1 8 , N O 1 0 , O C TO B E R 2 0 0 9

bacterial DNA targets) in the PCR reaction. In sim-
ple terms, a cycle refers to how many temperature
cycles it takes before the level of fluorescence of
the SYBR green passes a designated detection
threshold.
Results and discussion
Choosing correct bacterial concentration and
ratios for initial inoculation
Bacterial counts associated with the inoculums were
obtained in two different ways. For both methods,
optical density (OD) readings at 600nm were
obtained for comparison.
Traditional bacterial counts were collected by per-
forming serial dilutions to 1 x 102 bacteria on TSA
plates with a spiral plater. After 24 hours at 37C
colonies were counted and bacterial counts were
calculated based on the spiral plating protocols.
This was compared with the optical density read-
ings for reference.
Bacterial counts were also validated by perform-
ing a BacLight Viability test with fluorescence micro-
scopy. Once grown in culture, cells were stained
with a nucleic acid dye and filtered through a
0.22um black polycarbonate isopore membrane fil-
ter. Viable cells fluoresced green and dead cells fluo-
resced red. Viable cells were counted in 20 different
regions of the filter to obtain an overall average
count. Calculations for total bacterial number were
based off field of view of the microscope, amount
filtered, and total culture volume. Once again,
counts were compared with optical density readings
from culture for reference.
Both counting systems were similar in bacterial
cell count, and optical density readings for these
tests were logged as a reference for subsequent runs.
Bacterial cultures were diluted and added to a final
mixture that would yield consistent bacterial con-
centrations for each species added to the model.
Biofilms containing Staphylococcus aureus, Entero-
coccus faecalis and Pseudomonas aeruginosa were
grown and tested for 1:1:1 ratio for these three bac-
teria by inoculating 5 x 102 total bacteria into the
10ml model. Anaerobes for all tests were inoculated
a 2X concentration of the aerobic bacteria to evalu-
ate their growth along with the aerobes.
Examination of biofilms by electron microscopy
Ultrastructure evaluation of the LCWB was per-
formed using scanning (SEM) on biofilms contain-
ing Enterococcus faecalis and Staphylococcus aureus
(coccus) and Clostridium perfringens (rod) to visualise
morphological differences.
A complex inter-connected fibrous network of
extracellular polymeric substances (EPS), which play
a key role in biofilm formation, of differing thickness
was visible when using SEM. All three types of bacte-
rial cells were observed in the biofilm matrix (Fig 1).
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research
Clostridium perfringens was shown to propagate
within the biofilm, as confirmed by the presence of
rod-shaped bacteria within a biofilm that other-
wise only contained cocci-shaped bacteria (Entero-
coccus faecalis and Staphylococcus aureus). Aggrega-
tion of bacterial cells is common in chronic wound
pathogenic biofilms, and the cross-linking of the
bacterial cells with the EPS is an integral part of the
biofilm structure.
Population monitoring
To determine the bacterial composition of the bio-
films, qPCR was performed on the extracted DNA.
DNA extraction protocols can disrupt not only the
biofilm, but also lysing Gram-positive bacteria, which
are essential for accurate population monitoring. The
extraction methods described earlier have been test-
ed repeatedly and are efficient for extraction of Gram-
negative and Gram-positive bacteria.4,7
After extracted DNA was run through qPCR,
results were analysed as described earlier and bacte-
rial populations were determined. For all anaerobic
bacteria that showed integration, this proved to be
true. As mentioned earlier, this has been seen in
water sludge flocs, where a strict anaerobe Bacter-
oides spp., was detected in aerobically grown micro-
bial granules.5
However, our model represents the first rapid
screening wound biofilm model to show this phe-
nomenon. Of the five bacteria tested, some exhibit-
ed good integration into the biofilm, while others
Fig 1. Scanning electron micrographs of the Lubbock chronic wound biofilm
model with both aerobes and anaerobes
These micrographs illustrate the complex extracellular matrix created by the bacteria. Higher
resolution images show both rods (anaerobic bacteria, indicated with yellow arrows) and cocci
(aerobic bacteria, indicated with blue arrows), in close proximity, suggesting the cooperative
interaction of these communities
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Table 1. Population monitoring of anaerobic bacteria within an aerobic biofilm model.The

relative percentage of each population, including the anaerobes at 24 and 48 hour intervals,
are given
A P. aeruginosa E. faecalis S. aureus C. perfringens

24 hours 48 hours 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Ct 17.27 16.93 18.39 18.74 18.51 19.35 34.42 29.16

% bacteria 51.58% 66.33% 25.72% 19.44% 22.18% 14.23% 0.01% 0.5%

B P. aeruginosa E. faecalis S. aureus P. ivorii

24 hours 48 hours 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Count 18.26 17.12 18.11 17.57 18.53 19.67 31.16 20.58

% bacteria 33.61% 51.49% 38.45% 37.92% 27.93% 5.57% 0.5% 4.52%

C P. aeruginosa E. faecalis S. aureus P. anaerobius

24 hours 48 hours 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Ct 17.34 17.31 18.29 19.02 17.88 19.33 37.29 36.63

% bacteria 52.67% 64.30% 22.56% 18.53% 24.78% 11.05% 1.02% 5.10%

D P. aeruginosa E. faecalis S. aureus A. lactolyticus

24 hours 48 hours 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Ct 17.18 16.77 18.20 18.08 18.01 19.15 34.53 26.87

% bacteria 53.80% 61.10% 22.80% 25.71% 20.38% 13.16% 0.02% 3.00%

E P. aeruginosa E. faecalis S. aureus F. magna

24 hours 48 hours 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Ct 16.78 16.28 18.33 18.57 20.01 20.06 22.34 16.86

% bacteria 70.58% 55.23% 20.61% 7.50% 7.41% 1.96% 1.40% 35.31%

The Ct value refers to the threshold cycle

showed moderate integration (Table 1). Significant-
ly, the anaerobic bacteria not only incorporated
themselves into the matrix, but their bacterial pop-
ulation also increased dramatically, indicating that
they would reproduce within the biofilm.
Clostridium perfringens showed slight growth at 24
hours and integration after 48 hours growth (Table
1a). Clostridium perfringens is the most widely dis-
tributed pathogen in nature, but its prevalence in
our biofilm model is consistent with in vivo biofilm
wound types, where it is typically found in lower
percentages than other anaerobic bacteria.4,7
Peptoniphilus ivorii showed slight growth at 24
hours, but much higher abundance after 48 hours
(Table 1b). Although it was not as abundant as Fine-
goldia magna at 48 hours, a 4% ratio still correlates
to approximately 1 x108 bacteria. Peptoniphilus ivorii
has also been shown to be an important part of pol-
ymicrobial infections, especially DFUs and PUs.4,7
These multispecies wound types also included Sta-
phylococcus and Enterococcus species, lending validity
to our in vitro model. Peptostreptococcus anaerobius
showed integration at 24 hours and 48 hours incu-
bation time (Table 1c). It has been shown to be an
important anaerobic bacterium in wound patho-
genic biofilms such as leg ulcers.23,24
Anaerococcus lactolyticus showed slight growth at
24 hours and integrated at 48 hours (Table 1d).
Once again, this Gram-positive organism has been
shown to populate certain wound types including
PUs and DFUs and, in some cases, is one of the most
prevalent species in the microbial community.4,7
Like Peptoniphilus ivorii, Anaerococcus lactolyticus
shows growth in vivo in the presence of Staphyloco-
ccal species, supporting our use of these bacteria in
our model.

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 research

Finally, Finegoldia magna is a Gram-positive organ- Conclusion

ism and is part of the normal human flora of the
skin and mucous membranes. However, it has been
shown to be an opportunistic pathogen in several
clinically significant diseases, such as soft tissue
infections, septic arthritis, osteomyelitis and poly-
microbial infections.4,7,13,25
In our study, Finegoldia magna integrated into the
biofilm within 24 hours of inoculation, as shown by
qPCR. At 48 hours, it became the predominant spe-
cies, along with Pseudomonas aeruginosa (Table 1e).
This shows that Finegoldia magna may synergistical-
ly propagate along with the other bacteria, or can
survive until an anaerobic environment presents
itself through biofilm formation. With a starting
inoculum of ~1 x 103 colony forming units (CFUs),
all anaerobes were >1 x 106 CFUs/mg dry weight
biofilm after 48 hours. Finegoldia magna showed the
most dramatic increase over 48 hours within this
biofilm model, with an estimated concentration of
>1 x 108 CFUs/mg biofilm dry weight. This is a sur-
prising concentration within this aerobically incu-
bated biofilm, suggesting that this bacterium in par-
ticular will make a very good anaerobe model for
our polymicrobial in-vitro model for chronic wounds.
This is supported by documentation that suggests it
is ubiquitous within actual chronic wounds.
The development of population monitoring of clin-
ically significant anaerobes integrating into an aero-
bically grown biofilm, shows the functionality of
the LCWB as a rapid in-vitro method for the study of
the chronic wound biofilm paradigm.
Our media, which contains plasma and a founda-
tion derived from chopped meat (Bolton broth),
represents a logical nutritional base, which can
arguably simulate the nutrients available in a wound
environment.
The rapidity of biofilm development and matura-
tion, the ease of use, the tools described herein for
evaluation of the individual bacterial populations, a
heterogeneous composition, high reproducibility,
the use of plasma, and the relatively low cost of the
model provide advantages over current in vitro mod-
els for the study of chronic wound biofilms.
This model will facilitate the search for therapeu-
tics that can effectively target chronic wound
biofilms and other biofilm diseases. It is also now
being used to screen the next generation biofilm
effectors.
Work continues to be done to fine tune the ana-
lytical components and increase the functional and
species diversity of the bacterial populations within
the model.

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