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\begin{document}

\title{Review of the role of biofilms in chronic wounds in horses: clinical
indications and treatment strategies}



\author[1]{Kara Marchant}%
\author[1]{Dean Hendrickson}%
\author[1]{Lynn Pezzanite}%
\affil[1]{Colorado State University}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Recognition of the role that biofilms play in the persistence of chronic
wounds and lack of response to therapy in horses is increasing.
Prevention of biofilm development in early stages of wound care involves
three primary strategies: wound debridement and cleansing to reduce
bacterial counts in the wound bed, appropriate use of advanced wound
dressings, and implementation of topical antimicrobial agents. Once
formed, eradication of biofilms requires elimination to improve the
wound environment for contraction and epithelialization while not
further harming the native cells integral to the healing process, which
is achieved predominantly through repeated lavage and debridement
combined with topical antimicrobial therapy. This review will establish
why and how biofilms form, how to recognize clinical indications that
biofilms have formed in equine wounds, and to review current diagnostic
options and biofilm-based wound care (BBWC) strategies to eradicate
biofilms. Clinical scenarios for cases in which biofilms developed and
were successfully treated will be presented. This review will advance
practitioners' understanding of the presence and role of biofilms in
chronic wounds and provide an updated summary of recommended treatment
strategies.%
\end{abstract}%



\sloppy


\textbf{Title:} Review of the role of biofilms in chronic wounds in
horses: clinical indications and treatment strategies

\textbf{Running Title:} Role of biofilms in chronic wounds

\textbf{Authors:} Kara Marchant, Dean A. Hendrickson, and Lynn M.
Pezzanite

\textbf{Institutional Affiliation:} Department of Clinical Sciences,
College of Veterinary Medicine and Biomedical Sciences, Colorado State
University, 300 West Drake Road, Fort Collins, Colorado 80523, USA

\textbf{Corresponding Authors' Email:} lynn.pezzanite@colostate.edu and
dean.hendrickson@colostate.edu

\textbf{Summary}

Recognition of the role that biofilms play in the persistence of chronic
wounds and lack of response to therapy in horses is increasing.
Prevention of biofilm development in early stages of wound care involves
three primary strategies: wound debridement and cleansing to reduce
bacterial counts in the wound bed, appropriate use of advanced wound
dressings, and implementation of topical antimicrobial agents. Once
formed, eradication of biofilms requires elimination to improve the
wound environment for contraction and epithelialization while not
further harming the native cells integral to the healing process, which
is achieved predominantly through repeated lavage and debridement
combined with topical antimicrobial therapy. This review will establish
why and how biofilms form, how to recognize clinical indications that
biofilms have formed in equine wounds, and to review current diagnostic
options and biofilm-based wound care (BBWC) strategies to eradicate
biofilms. Clinical scenarios for cases in which biofilms developed and
were successfully treated will be presented. This review will advance
practitioners' understanding of the presence and role of biofilms in
chronic wounds and provide an updated summary of recommended treatment
strategies.

\textbf{Introduction}

Bacterial biofilms are organized communities of bacteria attached to a
surface and enveloped in a three-dimensional extracellular matrix. A
recent systematic review and meta-analysis of wound care literature in
humans reported the prevalence of biofilms in chronic wounds to be
78.2\% (Malone \emph{et al.,} 2017). Consensus guidelines for the
identification and treatment of biofilms have further stated that
biofilms should be assumed to be involved in most, if not all, chronic
non-healing wounds (Schulz \emph{et al.,} 2017). Furthermore, multiple
studies to date have documented evidence of biofilms in chronic wounds
of horses specifically (Freeman \emph{et al.,} 2009; Westgate \emph{et
al.,} 2011). The high prevalence of biofilms in non-healing wounds, the
frequency with which equine practitioners treat wounds in daily
practice, and the increasing reported incidence of multi-drug resistant
bacterial strains in equine practice in general (Herdan \emph{et
al.,}2012; Loncarac \emph{et al.,} 2014; Theelin \emph{et al.,} 2014;
van den Eede \emph{et al.,} 2012), highlight the need for implementation
of more advanced training in wound care strategies to address these
clinical scenarios.

The purpose of this review is to summarize the current literature
describing problems caused by bacterial biofilms in wounds, clinical
indications that biofilms are involved, laboratory testing to improve
biofilm detection, and biofilm-based wound-care (BBWC) strategies to
provide clinicians with practical guidelines for case management where
biofilms are suspected. Recommendations for antimicrobial duration in
veterinary practice further support administration for the shortest
effective duration to reduce risk of development of resistant pathogens
(Hansen \emph{et al.,} 2014; Gandini \emph{et al.,} 2022); therefore,
local surgical and topical techniques to address biofilm formation will
be emphasized to minimize unnecessary systemic antimicrobial
administration. Early recognition of the presence of biofilms in
non-healing wounds and targeted treatments are key to the successful
management of biofilms in equine practice (Pezzanite \emph{et
al.,}2021).

\textbf{Understanding the role of biofilms in wounds}

Biofilm formation is divided into three main stages: bacterial
attachment, growth, and detachment (Lappin-Scott and Bass 2001). In
stage one, planktonic (free-floating) bacteria adhere to surfaces within
several minutes (Parsek \emph{et al.,} 2005). In stage two, individual
attached bacteria (\emph{i.e.,} `sessile') secrete a three-dimensional
extracellular matrix (also known as extracellular polymeric substance
(EPS)) that includes water, proteins, glycolipids, polysaccharides,
bacterial DNA, and potentially other microbes benefiting from the
protected environment which makes up 90\% of the biomass of the biofilm
itself (Clutterbuck \emph{et al.,} 2007; Jacques \emph{et al.,} 2010;
Wolcott \emph{et al.,} 2008a; Percival, McCarty, Lipsky 2015; Flemming
and Wingender 2010). This occurs within 6 to 12 hours of attachment and
the biofilm continues to grow based on coordinated cell-to-cell
signaling known as `quorum-sensing.' (Parsek \emph{et al.,} 2005; Prada
\& S\selectlanguage{polish}ă\selectlanguage{english}ndulescu, 2019; McCarty \emph{et al.,} 2012). In stage three,
biofilms reach maturity within 2 to 4 days and shed free-floating
planktonic cells which disperse and attach to other areas of the wound
bed (Kostakioti, Hadjifrangiskou, \& Hultgren 2013). This cell
distribution activates the host immune response, which further
stimulates production of exudates that provide nutrients and promote
survival of the biofilm (Orsini \emph{et al.,} 2017; Dart \emph{et
al.,}2017a; Stewart and Richardson, 2019), and may lead to additional
complications for the host animal, including bacteremia or bacterial
colonization of distant anatomical sites (Bjarnsholt \emph{et.
al.,}2013).

Predisposing factors to biofilm formation include the presence of
foreign bodies, sequestra and surgical implants, reduced vascular
perfusion to the anatomical region, inappropriate antimicrobial
sensitivity, and the immune status of the patient (age, sepsis,
malnutrition, antibody deficiency, chronic stress, corticosteroid
administration, or underlying diseases including pituitary pars
intermedia dysfunction {[}PPID{]} or Cushing's disease) (Seth \emph{et
al.,} 2012, Orsini \emph{et al.,} 2017). Strategies to prevent biofilm
development in acute wounds include wound debridement and cleansing to
reduce bacterial counts and appropriate use of advanced dressings and
topical antimicrobial agents. Addressing systemic conditions
(\emph{e.g.,} Cushing's disease in horses) may promote more rapid
bacterial clearance and healing in immune-incompetent patients as well.
Furthermore, the ability of the host's immune response to effectively
control microbes decreases as the biofilm matures. As a consequence,
infections involving biofilms frequently recur following discontinuation
of antimicrobials (Dart \emph{et al.,} 2017b), emphasizing that early
recognition of treatment of both the wound and the animal's systemic
health status are key to successful management.

Wounds with biofilms may not necessarily exhibit signs typically
associated with infection besides prolonged and impaired healing
(Dart\emph{et al.,} 2017a). The presence of biofilms has been
demonstrated to delay epithelialization and induce a chronic non-healing
inflammatory state (Wolcott \emph{et al.,} 2008; Schierle \emph{et al.,}
2009). However, it is important to note that polymicrobial biofilms,
which are considered more pathogenic than monobacterial colonies, have
been reported in multiple types of equine wounds, not limited to those
considered chronic (\emph{e.g.,} acute or chronic, surgical or traumatic
in origin) (Westgate \emph{et al.,} 2001; Freeman \emph{et al.,} 2009;
Pastar \emph{et al.,} 2013). Metabolically active, nondividing persister
cells, which are tolerant to antimicrobials, are integral to
reestablishing biofilms following topical treatments (Kostakioti,
Hadifrangiskou and Hultgen, 2003). Specific bacterial species may
integrate chromosomal \selectlanguage{greek}β-\selectlanguage{english}lactamase, efflux pumps, and mutations in target
antibiotic molecules to evade host defenses. Finally, extracellular DNA
(eDNA) present in bacterial biofilms promotes acid-base interactions
between bacterial cells and surfaces, therefore playing an essential
structural role in both establishing biofilms and protecting cells
within the biofilm from environmental challenges (Lewenza \emph{et
al.,}2013; Thomann \emph{et. al.,} 2016).

Locally, polymicrobial infections delay wound closure through alteration
of cytokine levels and receptors (Pastar \emph{et al.,} 2013). For
example, \emph{S. aureus} and \emph{P. aeruginosa} are known to
downregulate keratinocyte growth factor 1 expression of fibroblasts,
resulting in delayed re-epithelialization through reduction of
keratinocyte migration and proliferation (Pastar \emph{et. al.,} 2013).
Bacteria in biofilms secrete enzymes (\emph{e.g.,} proteases, elastase,
phospholipase) to degrade local host tissues to provide nutrients and to
protect bacteria within the biofilm from host immune cells
(Michalkiewicz \emph{et al.,} 1999; Flemming and Wingender, 2010;
McCarty \emph{et al.,} 2017). For example, the proteases secreted
by\emph{Pseudomonas aeruginosa} degrades and inactivates interferon
gamma which suppresses innate immune recruitment and reduces elimination
of biofilm bacteria (Michalkiewicz \emph{et al.,} 1999). Continuous
production of exudate is detrimental to wound healing as the
inflammatory process continuously breaks down the ECM (McCarty \emph{et.
al.,} 2012) and may degrade growth factors associated with normal wound
healing processes (Percival \emph{et al.,} 2015). Various cell types
including keratinocytes, fibroblasts, endothelial cells, and
inflammatory cells (\emph{e.g.,} monocytes, lymphocytes, and
macrophages) express matrix metalloproteinases (MMPs) involved in
epithelial repair, wound contraction, and degradation of damaged ECM
within the skin (Caley \emph{et al.,} 2015) which is upregulated in
wound edge keratinocytes to allow epidermal cell migration across wound
beds (McCarty \emph{et al.} 2012). However, in wounds associated with
biofilms, the presence of devitalized tissue and abnormal immune cell
activity results in excessive production of MMPs which perpetuates ECM
destruction propogating the inflammatory response and wound chronicity
(Caley \emph{et al.,} 2015; Parnham and Bousfield 2018; Kandhwal\emph{et
al.,} 2022). Approaches to restore normal wound healing involve
techniques directed towards inhibition of these biofilm virulence
factors through effective, sustained debridement of devitalized tissues
(Schierle~\emph{et al.} , 2009; Parnharm and Bousfield 2018).

Development of infection involving biofilms has important implications
in management of wounds in horses, as they present unique challenges in
diagnosis and are more resistant to typical treatment methods
(Dart\emph{et al.,} 2017a). Bacteria that produce biofilms are able to
survive and grow at slower metabolic rates in environments depleted of
nutrients and oxygen, termed phenotypic heterogeneity (Donlan \emph{et
al.,} 2001; Clutterbuck \emph{et al.,} 2007). Mature biofilms secrete
protective enzymes, shielding themselves from host defenses and exterior
physiologic changes that may be detrimental to bacterial health
(Percival \emph{et al.,} 2015). Once formed, bacteria in biofilms
differentiate into complex communities with enhanced resistance to
environmental challenges (\emph{e.g.,} cells of the innate immune
system, desiccation, etc.), biocides, and antibiotics (Costerton\emph{et
al.,} 1999; Fux \emph{et al.,} 2005) and variable morphology depending
on nutrient availability (Klausen \emph{et al.,} 2003, Flemming and
Wingender, 2010). As a result, bacteria within biofilms are more
tolerant to the host immune response, antimicrobial therapy administered
systemically (antibiotics) or topically (antiseptics) including hydrogen
peroxide, alcohols, bleach, oxygen radical generators and acids (unless
administered at concentrations toxic to the animal's cells) (Clutterbuck
\emph{et al.,} 2007). For example,\emph{Staphylococcus aureus} has been
shown to be up to 100 times more resistant to antimicrobials when in
biofilm versus planktonic form (Leid\emph{et al.,} 2002). These
challenges in addressing bacteria in biofilms may only be overcome if
antimicrobials to which the bacteria are sensitive can be delivered at
adequate concentrations for a sufficient time to achieve bactericidal
activity (Stewart and Richardson, 2019).

\textbf{Diagnosing biofilms -- laboratory testing and clinical
indications}

Traditional bacterial culturing techniques are generally considered
inadequate to comprehensively identify bacterial species associated with
biofilms (Kirketerp-Moller \emph{et al.,} 2008). Diagnosis of biofilms
in wounds can only be definitively made using scanning electron or
confocal microscopy imaging or molecular techniques to identify
bacterial components, which are not readily available modalities to
clinicians (Wolcott and Rhoads 2008; Percival \emph{et al.,} 2015;
Dart\emph{et al.,} 2017; Schulz \emph{et al.,} 2017; Hurlow \emph{et
al.,}2015). Recent studies have demonstrated that biofilms associated
with wounds are most commonly polymicrobial communities, with an average
number of 3.02 +/- 1.65 species identified (range, 0-8)
(Westgate\emph{et al.,} 2001; Freeman et al. 2009). Genera identified
were similar to those found in human infections, with \emph{Pseudomonas,
Enterococcus} , and \emph{Staphylococcus} species being most common
(Wolcott and Rhoads 2008; James \emph{et al.,} 2008; Dowd \emph{et
al.,}2008; Darvishi \emph{et. al.,} 2021). However, molecular analyses
of chronic wound samples have revealed far more diverse polymicrobial
communities with up to 17 genera per wound, including anaerobic species
not identified by routine culturing, and further highlighting the
challenges faced by clinicians in accurately identifying and treating
bacterial species contained within biofilms (James \emph{et al.,} 2008;
Han \emph{et al.,} 2011).

Standard methods to assess bacterial burden in wounds include
qualitative and quantitative techniques (Hendrickson 2019a). Qualitative
assessment determines the genera of bacteria found in wounds and is
coupled with sensitivity testing to provide clinicians basis for
antibiotic choices in treatment. Quantitative bacteriology methods are
less commonly performed in veterinary medicine but should be considered
in cases when wound healing is not progressing as anticipated or
following skin graft failure. Active infection has typically been
considered to be the case in situations where bacterial counts are found
to be greater than 10\textsuperscript{5} per gram tissue or mL exudate
(Robson and Heggers, 1969). However, the number of bacteria required to
establish an infection is reduced in situations where the patient's
bacterial resistance or immunocompetence is decreased, foreign material
is present including implants, sutures, foreign bodies or necrotic
debris, or bacterial virulence is high (Bowler 2003). In polymicrobial
infections, as is most typical of those involving biofilms, multiple
microorganisms act synergistically to result in greater virulence
compared to an infection caused by either species alone (Serra \emph{et
al.,} 2015). In cases involving multidrug resistant isolates, as few as
100 bacteria per gram tissue or mL exudate may be sufficient to incite
infection (Rodeheaver \emph{et al.,} 1974).

The best diagnostic method currently available to clinicians in equine
practice when biofilms are suspected is submission of a deep tissue
biopsy or swab of the deepest tissues available (or both) for bacterial
culture and sensitivity to guide future treatment practices
(Dart\emph{et al.,} 2017). In general, tissue samples, while being more
invasive to collect, are more likely to yield reliable culture results
compared to swabs (Westgate \emph{et al.,} 2001; Freeman \emph{et
al.,}2009). Ideally, submission of tissue samples should be performed
prior to beginning or altering antimicrobial protocols; however, if
considered necessary to collect samples while horses are currently
receiving antimicrobial, it is recommended to notify the receiving
laboratory of the horse's current regimen and when the most recent dose
was received in relation to sample collection (Orsini \emph{et al.,}
2017). Following superficial wound debridement, tissue samples should be
collected from within the deepest regions of the wound (\emph{e.g.,}
fissures or pockets in the wound bed) and from multiple sites if
possible to avoid false positive results (Sen \emph{et al.,} 2012;
Rhoads \emph{et al.,}2012). If tissue swabs are collected, the swab
should be drawn across the wound surface with sufficient pressure to
collect the biofilm itself while avoiding drawing blood which contains
antimicrobial elements that may affect culture results. Positive culture
results should be interpreted with the assumption that the full
microbial spectrum is likely underrepresented with currently available
techniques.

In lieu of obtaining a positive culture result or if submission is not
an option due to financial or other case-related considerations,
diagnosis of biofilms in wounds may be based on clinical indications
(\textbf{Table 1} ). Clinical findings consistent with biofilm presence
include indicators of inflammation (heat, swelling, pain, redness),
persistent or recurrent infection despite administration of
antimicrobial therapy or recurrence following antibiotic
discontinuation, excessive wound moisture/exudate, poor quality
granulation tissue, history of negative culture findings despite
clinical suspicion of infection, or in general a wound that remains in a
chronic and recalcitrant inflammatory state despite standard treatment
and evaluation of the patient for comorbidities
(\emph{e.g.,}immunosuppression). In summary, culture findings to
diagnose biofilms are unreliable and observation of clinical indications
that biofilms are present in the wound bed should prompt practitioners
to implement wound care strategies directed specifically at addressing
and reducing biofilm formation in wounds.

\textbf{Biofilm-based wound-care treatment strategies}

4.1. Biofilm-based wound-care guidelines - Recent consensus documents in
human wound care have described biofilm-based wound-care (BBWC)
strategies to provide practical guidelines for case management in which
biofilms are suspected (Wolcott and Rhoads, 2008b; Schultz \emph{et
al.,} 2017; Metcalf \emph{et al.,} 2014; Bianchi\emph{et al.,} 2016)
(\textbf{Table 2} ). Biofilm treatment is recommended in three stages:
1) physical debridement of the biofilm, 2) topical treatment to delay or
prevent reformation, and 3) repeated therapy until full resolution is
achieved (Orsini \emph{et al.,} 2017). These strategies emphasize that
repeated debridement to physically disturb the biofilm structure is
necessary to disrupt the matrix and remove devitalized tissues that
serve as nutrients to the microbes involved and allow increased
susceptibility to antimicrobial therapies for a period of time to
prevent bacterial reattachment as immature biofilms are more susceptible
to antimicrobials (Dart \emph{et al.,}2017). Implementation of a
multimodal therapeutic strategy to address biofilms has a reportedly
higher success rate compared to antimicrobials alone (Wu \emph{et al.,}
2015).

4.2. Debridement principles - The overall objective of debridement is to
remove as much devitalized tissue, biofilm and associated extracellular
matrix as possible to expose the remaining bacteria to antimicrobial
agents. The organization and complex physiology of mature biofilms
increases their resistance to antibiotics resulting in colonized
bacteria being up to 1000-fold times more resistant to antimicrobials
than planktonic cells (Hoiby \emph{et al.,}2010). Debridement removes
ECM and eDNA to prevent recurrence of biofilms in the wound by removing
the basis for nutrition and protection of the bacterial component of the
biofilm (Hajska \emph{et. al,} 2014). The immature biofilms that begin
to reform following debridement are subsequently more susceptible to
topical therapies. General principles described by Wolcott \emph{et al.}
in addressing wounds infected with biofilms include debridement with the
goal to alter the wound bed anatomy by removing any devitalized or
discolored tissue and all tissue surfaces that touch one another until
normal bleeding tissue is encountered (Wolcott \emph{et al.,} 2010).
Application of topical treatments is then recommended within four hours
following debridement prior to biofilm reformation (Roche \emph{et al.,}
2012; Hajska \emph{et al.,} 2014). An example of how biofilms may be
successfully treated and how rapidly they reform in the absence of
consistent treatment is daily removal of dental enamel plaque by regular
tooth brushing (i.e., debridement) performed in combination with topical
antiseptic mouthwashes, which are of minimal benefit without prior
flossing and tooth brushing (Orsini \emph{et al.,} 2017).

Biofilm debridement may be performed sharply (\emph{e.g.,} scalpel
blade), mechanically with gauze across the wound bed, or using water-jet
irrigation or low-frequency ultrasonic debridement. It is recommended
that horses be sedated, and the wound desensitized with local or
regional anesthesia to facilitate procedures and reduce discomfort to
the patient. In some cases, the initial debridement may be performed
under general anesthesia if the wound is extensive or inaccessible or if
dictated by the patient's temperament. When working with multi-drug
resistant organisms or particularly when using pulsed water-jet
irrigation, face protection or use of surgical masks during the
debridement stage is recommended to protect again aerosolized organisms.
Debridement and efforts to reduce biofilm reconstitution should be
repeated daily to at least every other day for as long as necessary to
resolve infection. Mature biofilms reform as rapidly as every 24 to 72
hours after debridement, resulting in a window of opportunity to impede
regrowth in which topical therapies and bactericidal drugs can exert an
enhanced effect. If improvement is not observed within three to four
days of initiation of the multimodal therapeutic approach outlined or if
response to therapy is less than anticipated, review of all aspects of
the case is indicated. These may include repeated physical examination,
bloodwork, evaluation of antibiotic suitability with repeated bacterial
culture and sensitivity, and further debridement and exploration of the
wound and potentially additional diagnostic imaging to evaluate for
alternate reasons for delayed healing (\emph{e.g.,} foreign material).

4.3. Topical therapies to prevent biofilm reformation - Reduction or
prevention of biofilm reformation following debridement may be achieved
in multiple ways. Topical antiseptic agents do not penetrate necrotic
debris and have minimal effect to reduce bacterial populations deep in
the wound bed or without debridement; therefore, they should generally
be reserved for use on intact skin and in wound beds (Alves\emph{et
al.,} 2021). Examples of antiseptic agents contraindicated for use in
biofilm associated wounds include alcohols, hydrogen peroxide, iodine,
povidine-iodine, chlorhexidine, aluminum salts, boric acid,
formaldehyde, hexachlorophene, hypoclorite, merthiolate, or
permanganate. However, unlike antiseptics, topical antimicrobial agents
can have minimal negative side effects on wound healing depending on the
vehicle and dose used and provide efficacy against bacteria in the wound
bed when administered following debridement and based on results of
culture and sensitivity.

Surfactant dressings such as polyhexamethylene biguanide (PHMB) or
polyhexanide can be used as adjunctive therapies in the early
post-debridement period, as they reduce biofilm surface tension to
facilitate degradation and removal (Palumbo \emph{et. al.,} 2016;
Percival \emph{et al.,} 2019). Other topical dressings such as silver
sulfadiazine (1\%) or other silver impregnated wound dressings may be
used in the early post-debridement stage to reduce biofilm reformation,
particularly if bacterial culture and sensitivities to guide topical
antimicrobial treatments are not available (Morones \emph{et al.,} 2005;
Fey \emph{et al.,} 2010; Gunaskaran \emph{et al.,} 2012). Silver works
through interacting with ribosomes to suppress enzymatic expression and
protein formation essential for ATP production (Yamanaka \emph{et
al.,}2005). The methods by which silver interacts with bacteria reduces
formation of resistance and results in broad-spectrum antibacterial
properties (Gunaskaran \emph{et al.,} 2012). In addition, silver
enhances re-epithelialization, angiogenesis, deposition of collagen
fibers, and myofibroblast distinction from fibroblasts prompting wound
contraction (Toczek \emph{et. al.,} 2022). Manuka honey also exhibits
antimicrobial properties due to high methylglyoxal and leptosperin
content and may be used as an adjunctive topical antimicrobial therapy
against a variety of bacterial species with minimal host cytotoxicity
(Molan and Rhodes, 2015; Liu \emph{et al.,} 2017). Finally, topical
application of plasma (autologous natural plasma or hyperimmune plasma
to target specific organisms) may provide additional benefit as a
topical therapy as plasma inhibits bacterial adhesion and growth
(Felts\emph{et al.,} 2000; Bauer \emph{et al.,} 2004; Lopez \emph{et
al.,}2014).

4.4. Antimicrobial guidelines -- In general, contaminated wounds
including those with suspected biofilm involvement are more
appropriately treated with bactericidal versus bacteriostatic
antimicrobial agents. Although ideally dictated by culture and
sensitivity findings, broad-spectrum antimicrobial therapy is generally
instituted initially with commonly administered agents include
penicillins, cephalosporins, aminoglycosides, quinolones, metronidazole
and rifampin (Orsini \emph{et al.,} 2017). Commonly used initial
combinations include pencillin G (crystalline or procaine penicillin) or
a cephalosporin (cefazolin or ceftiofur) and an aminoglycoside such as
gentamicin. Collection of a separate sample to perform in-house
point-of-care Gram staining may help to guide interim antimicrobial
therapy in lieu of culture and sensitivity findings. In treatment of
distal limb wounds, antimicrobials can also be delivered via regional
limb perfusion. Antimicrobial concentrations delivered locally are
greatest immediately following biofilm degradation so timing of
perfusion to directly follow debridement may improve outcomes although
further investigation is indicated. Finally, repeated culture and
sensitivity is also generally considered indicated in cases where
response is less than anticipated, signs of infection recur following
discontinuation of antimicrobials, if the infection is polymicrobial or
multidrug resistant, or during periods of prolonged antimicrobial
administration.

4.5. Other considerations - Limitations of current laboratory testing
and definitive clinical signs indicating biofilm presence make it
impossible to objectively determine whether biofilms have been
eradicated from a wound. Further investigation of stall-side testing
techniques to identify biofilm presence may enhance monitoring
techniques in the future; however, currently, monitoring of clinical
progression with reduced exudate and slough remains the most effective
method to determine response to treatment and biofilm resolution
(Leaper\emph{et al.,} 2012). However, despite appropriate treatment,
biofilms associated with orthopedic implants or other foreign devices
frequently necessitate removal for resolution (Richardson and Stewart
2019). In some cases, infection can be controlled temporarily through a
combination of systemic and local antimicrobial therapy until fracture
or arthrodesis consolidation has occurred (Wu \emph{et al., 2014} ). If
cases with both infection and instability, implants may be removed and
replaced, or cleaned, sonicated, and reimplanted using new orthopedic
screws when financially feasible. Alternatively, internal implants may
be removed, and cases managed with a transfixation pin cast or other
external fixator. The fracture site and surrounding tissues should be
debrided and lavaged, and previous screw holes and the surrounding
region may be treated locally with antibiotic eluting materials. In
general, when communicating with clients about the cost of care in
biofilm-associated wounds, it is recommended to emphasize that the
greater expense incurred in the earlier stages of wound management
typically reduces duration of therapy and costs overall in treatment
long-term (Orsini \emph{et al.,} 2017). Clinical case examples where
wounds with biofilms were successfully treated are provided and
summarized in \textbf{Figures 1 and 2} .

\textbf{Future directions in diagnostic techniques and treatment
strategies for biofilms}

Novel techniques to reduce infection burden associated with biofilms are
currently being investigated and further developed. Methods described
include further investigation of surfactant-based agents, cellular
therapeutic options (\emph{e.g.,} platelet rich plasma lysates,
mesenchymal stromal cells), quorum-sensing inhibitors (RNAIII inhibiting
peptide), hydrophobic polycationic or sol gel coatings, bacteriophage
therapies (antibacterial viruses), antimicrobial peptides, ultraviolet
light, low-voltage pulsed electrical fields, acetylsalicylic acid,
xylitol, dispersin B, gallium, or antimicrobial tethering
(Tiller\emph{et al.,} 2001; Levy \emph{et al.,} 2004; Nablo \emph{et
al.,}2005; Balaban \emph{et al.,} 2005; Williams and Hare 2011;
Stewart\emph{et al.,} 2012; Schaer \emph{et al.,} 2012; Barsotti
\emph{et al.,}2013; Spaas \emph{et al.,} 2013; Bussche \emph{et al.,}
2015; Mohammed\emph{et al.,} 2016; Grassi \emph{et al.,} 2017; Orsini
\emph{et al.,}2017; Hans \emph{et al.,} 2019; Gilbertie \emph{et al.,}
2021; Gordon\emph{et al.,} 2021). In addition, future diagnostic tests
may be more effective at definitively identifying the presence and
location of biofilms within a wound bed to guide more patient-specific
treatment strategies. For example, a stall-side or patient-side test to
quantify wound bed protease activity could be one method to indirectly
quantify and longitudinally evaluate the amount of residual biofilm in a
wound, as protease activity correlates generally to the amount of viable
or active biofilm (Leid \emph{et al.,} 2002). Further evaluation of
methods to improve detection of biofilms, monitoring of treatment
efficacy, and overall management of biofilms in case-controlled studies
and randomized controlled clinical trials in warranted.

\textbf{Conclusions}

The recognition that most chronic wounds in equine practice involve
pathogenic bacterial biofilms is key to successful treatment. Clinical
indications that biofilms are present in the wound bed include wounds
that remain in a chronic inflammatory state recalcitrant to standard
therapies, excessive exudate/moisture, poor quality local granulation
tissue, other common indications of infection (heat, swelling, pain),
and/or negative bacterial culture results despite clinical suspicion of
infection. Biofilm-based wound care strategies emphasize repeated
debridement and lavage combined with topical surfactants or
antimicrobials applied within four hours of debridement that have
minimal local cytotoxicity to host tissues. Finally, improved diagnostic
tools to detect biofilms and monitor response to treatment as well as
adjunctive treatments may facilitate improved outcomes in the future.

\textbf{Authors' declaration of interests:} No conflicts of interest
have been declared.

\textbf{Ethical animal research:} Review of current literature; not
applicable.

\textbf{Source of funding:} None.

\textbf{Authorship:} All authors contributed to conception and design,
acquisition of data, drafting and final approval of the manuscript.

\textbf{Tables}

\textbf{Table 1:} Indirect clinical indications of wound biofilm.
(Metcalf et al, 2014; Wolcott et al. 2008)\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Clinical Observation} & \textbf{Biofilm Explanations for
Clinical Observation}\tabularnewline
\midrule
\endhead
Excessive moisture associated with wound & Bacteria in biofilms secrete
extracellular matrix and biofilm presence promotes inflammation,
resulting in increased exudate.\tabularnewline
Autograft or allograft fails on wounds & Applying tissue grafts over
biofilms provides a second growth surface and food source, leading to
devitalization of graft tissue and increased exudate and
inflammation.\tabularnewline
Poor quality granulation tissue (e.g. hypergranular, friable) & Biofilm
presence contributes to delayed epithelialization and is frequently
associated with poor quality granulation tissue.\tabularnewline
Indications of local infection (swelling, sensitivity, redness, heat) &
Biofilms promote inflammation and may be a precursor to other clinical
indications of infection.\tabularnewline
History of persistent or recurrent infection despite antimicrobial
therapy & Biofilm bacterial phenotypes adapt rapidly and may only
demonstrate 1 to 2 log reduction with antibiotic therapy at 50 to 1000x
MIC. Biofilms contain persister cells that remain once antibiotic
therapy is discontinued, seeding, and contributing to subsequent biofilm
reformation.\tabularnewline
Negative culture results despite clinical suspicion of infection or
signs of bacterial colonization & Biofilm bacteria metabolize more
slowly and are phenotypically different than planktonic bacteria.
Standard microbiological culture techniques are not capable of
identifying all species present, making bacteria in biofilms difficult
or impossible to identify by culture.\tabularnewline
Wound remains in chronic inflammatory state and recalcitrant to therapy
despite addressing comorbities & Biofilms are resistant to host
inflammatory responses and actually feed off exudate produced by
inflammation, further promoting inflammation.\tabularnewline
\bottomrule
\end{longtable}

Republished with permission from \emph{Am Assoc Equine
Pract}2021;67:58-65. Copyright 2021 American Association of Equine
Practitioners.

\textbf{Table 2:} Key statements on biofilms in wounds (Schulz et al,
2017)\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Biofilms are present in most chronic wounds and are likely to be present
on both the surface and deeper wound layers but may not be uniform
across or within the wound.\tabularnewline
\midrule
\endhead
Biofilms are difficult to visualize macroscopically, and exudate, slough
or debris may be mistaken for biofilms.\tabularnewline
Wounds that contain biofilms may not be identified, leading to
ineffective treatment and delayed healing.\tabularnewline
Important clinical indications that a wound likely contains a biofilm
include lack of response to treatment with antibiotics or
antiseptics.\tabularnewline
Debridement is one of the most important treatment strategies against
biofilms; however, biofilms reform rapidly, so debridement should be
used in conjunction with topical antiseptics and
surfactants.\tabularnewline
\bottomrule
\end{longtable}

Republished with permission from \emph{Am Assoc Equine
Pract}2021;67:58-65. Copyright 2021 American Association of Equine
Practitioners.

\textbf{Figures}

\textbf{Figure 1: Case example 1.} 7-day old Warmblood foal was
presented for acute swelling of the right tarsus. An approximately 2cm x
2cm abrasion was noted over the lateral aspect of the tarsus with
moderate periarticular edema was appreciated. Radiographs of the tarsus
revealed no significant abnormal findings and fluid analysis of synovial
fluid from the tibiotarsal joint was within normal limits. Ultrasound of
the umbilicus, abdomen and thorax revealed no significant abnormal
findings. Within 3 days following presentation, the colt developed
marked cellulitis of the right hindlimb which was initially with a
compression/sweat bandage, and intravenous antibiotic therapy (amikacin,
potassium penicillin) and anti-inflammatories (flunixin meglumine).
Ultrasound of the limb revealed a suspected extra-articular subcutaneous
abscess forming near the level of the distal intertarsal and
tarsometatarsal joints. Five days after initial presentation,
strike-through was noted diffusely throughout the bandaged limb and when
the bandage was removed a generalized necrotic open wound extending from
the level of the tarsus to the fetlock was appreciated. Culture and
sensitivity yielded \emph{Citrobacter, Enterobacter}
and\emph{Staphylococcus aureus} . Based on sensitivity results,
antibiotics were transitioned to ceftiofur sodium. Repeated daily to
every other day topical debridement, lavage, and wound care with kerlix
AMD surfactant impregnated gauze dressings were performed. Approximately
3 weeks after initial presentation, the wound bed was deemed healthy
enough for skin grafting. Full thickness mesh graft obtained from the
ventral abdomen was performed under general anesthesia. Two weeks later
he received a second mesh graft to cover the remaining area of the
wound, obtained from his pectoral region. The tissue here obtained from
the ventral abdomen was a different color but overall, the result was
considered a good cosmetic outcome. The foal was dismissed to the care
of his owners after approximately 5 weeks hospitalization and continued
to do well at home with approximately four years follow-up to date.

\textbf{Figure 2: Case example 2.} A 14-year-old Quarter Horse gelding
was presented for evaluation of a 5-month-old wound on the dorsal aspect
of the tarsus, sustained the previous summer on barbed wire fence. He
had been initially treated with trimethoprim sulfamethoxazole
antibiotics. Radiographs of the tarsus were performed which revealed no
significant abnormal findings and he was turned out on pasture. When the
wound continued not to heal, he was brought to Colorado State University
Veterinary Teaching Hospital for further evaluation. Radiographs at that
time revealed no sequestrum or osseous involvement. The gelding was
induced under general anesthesia and the wound was sharply debrided and
lavaged. He was maintained on antibiotics with bandaging in kerlix AMD
and splinting to minimize motion through the tibiotarsal joint. A second
debridement was performed approximately 3 weeks following the first and
samples collected for culture. He was transitioned from trimethoprim
sulfamethoxazole antibiotics to enrofloxacin based on culture and
sensitivity results. Additionally, he was enrolled as a pilot case in a
study evaluating the effect of mesenchymal stromal cell therapy to
improve wound healing. The lateral half of the wound bed was treated
with 3 doses of intralesional doses of 30 million stem cells. At
approximately 3 months following initial presentation, the wound bed was
considered healthy enough to support a graft. A pinch skin graft was
performed from skin obtained from the left ventral abdomen.
Interestingly, although the wound did fully epithelialize by 10 months
following initial presentation, the lateral aspect of the wound that had
been injected with stromal cells did so more rapidly, indicating that
the antimicrobial and immunomodulatory properties of MSC may help to
accelerate healing and warrants further investigation.

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