Microb Ecol

DOI 10.1007/s00248-016-0867-9

REVIEW

Bacterial Contribution in Chronicity of Wounds

Kashif Rahim 1 & Shamim Saleha 2 & Xudong Zhu 1 & Liang Huo 1 & Abdul Basit 3 &
Octavio Luiz Franco 4,5

Received: 8 August 2016 / Accepted: 21 September 2016

# Springer Science+Business Media New York 2016

Abstract A wound is damage of a tissue usually caused by
laceration of a membrane, generally the skin. Wound healing
is accomplished in three stages in healthy individuals, includ-
ing inflammatory, proliferative, and remodeling stages.
Healing of wounds normally starts from the inflammatory
phase and ends up in the remodeling phase, but chronic
wounds remain in an inflammatory stage and do not show
progression due to some specific reasons. Chronic wounds
are classified in different categories, such as diabetic foot ulcer
(DFU), venous leg ulcers (VLU) and pressure ulcer (PU),
surgical site infection (SSI), abscess, or trauma ulcers.
Globally, the incidence rate of DFU is 1–4 % and prevalence
rate is 5.3–10.5 %. However, colonization of pathogenic bac-
teria at the wound site is associated with wound chronicity.
Most chronic wounds contain more than one bacterial species
and produce a synergetic effect that results in previously non-
virulent bacterial species becoming virulent and causing dam-
age to the host. While investigating bacterial diversity in
chron ic wou nds, Staphy lo coccus , Pseud omo nas,
Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia,
and Serratia were found most frequently in chronic wounds.
Recently, it has been observed that bacteria in chronic wounds
develop biofilms that contribute to a delay in healing. In a
mature biofilm, bacteria grow slowly due to deficiency of
nutrients that results in the resistance of bacteria to antibiotics.
The present review reflects the reasons why acute wounds
become chronic. Interesting findings include the bacterial
load, which forms biofilms and shows high-level resistance
toward antibiotics, which is a threat to human health in general
and particularly to some patients who have acute wounds.
Keywords Wound . Chronic wounds . Biofilms .
Resistance . Antibiotics
Introduction

A wound is damage of a tissue, caused by a burn, bone frac-

* Octavio Luiz Franco ture, skin cut, or muscle punctures. The cause of a wound may
ocfranco@gmail.com be unintentional, such as an animal bite or accident fall [1] or
intentional, such as gun shots and surgical interventions [2].
1
Institute of Biochemistry and Molecular Biology, College of Life The discontinuity of the skin at such sites allows organisms
Sciences, Beijing Normal University, Beijing 100875, China
and other foreign bodies to gain access into the tissues [3, 4] or
2
Department of Biotechnology and Genetic Engineering, Kohat the internal organs. Proper management of wounds is there-
University of Science and Technology (KUST), Khyber
Pakhtunkhwa Kohat 26000, Pakistan
fore vital for timely healing. When the skin becomes compro-
3
mised, the normal wound healing process resolves the injury.
College of Biological Sciences, China Agricultural University,
Beijing 100193, China
In order to understand why an acute wound could end up
4
being a chronic wound, the process of wound healing needs
Centro de Análises Proteômicas e Bioquímicas, Programa de
Pós-Graduação em Ciências Genômicas e Biotecnologia,
examination. Understanding the wound healing process will
Universidade Católica de Brasília, Brazil 70790-160, Brazil provide insight for evaluating the stage at which healing stops
5
S-Inova Biotech, Programa de Pós-Graduação em Biotecnologia,
and the factor that may have contributed to the stalling pro-
Universidade Católica Dom Bosco, Campo Grande CEP 79.117-900, cess. Wound healing is a repair process of damaged tissues
Brazil and organs in the human body. This repair is accomplished in

three stages in healthy individuals, including inflammatory,
proliferative, and remodeling stages [5] (Fig. 1).
There are many types of wounds which could be chronic.
In this review, we discuss some major chronic wounds such as
diabetic foot ulcers (DFU), venous leg ulcers (VLU) and pres-
sure ulcers (PU), surgical site infection (SSI), abscesses, or
trauma ulcers (Fig. 2). Nowadays, the basic issue is for dia-
betic patients, as almost 90 % of individuals with diabetic
issues and lower extremity amputation (LEA) have histories
of foot ulcers or have lower extremity findings consistent with
peripheral arterial disease (PAD) and/or neuropathy [6].
Moreover, the leading cause of morbidity and mortality are
because of hospitalization (long time stay) of DFU Patients
as compared to other complication in patients [7]. The second
type of chronic wound is venous leg ulcers, which are
present in patients who have experienced chronic venous in-
sufficiency; these are one of the most common chronic
Fig. 1 Schematic diagram of
wound types, healing process, and
prevalence of chronic wounds
K. Rahim et al.
wounds found in clinical practice. It is estimated that approx-
imately 1 million patients in the USA currently have this con-
dition [8]. The current standard clinical approach to therapy
includes aggressive compression of the lower limb with de-
bridement of the ulcer, which heals 50 to 60 % of venous leg
ulcers [9]. Wounds that are infected by microbiota after sur-
gery are known as surgical site infections, and this type of
infections may be due to the instruments and unsterilized en-
vironmental condition during or after the surgery. The infec-
tion may be superficial, deep, or covering the space between
two cut regions. In 2006, a prevalence survey was conducted
to find the rate of surgical site infections, and approximately
8 % of affected patients in the UK were found to have
healthcare-associated infections. SSIs accounted for 14 % of
these infections, and nearly 5 % of patients who had under-
gone a surgical procedure were found to have developed an
SSI, but most of these infections are after discharge from
Bacterial Contribution in Chronicity of Wounds
hospital [10]. Pressure ulcers are also a common problem
among older adults in all health care. Activity or mobility
limitation, incontinence, abnormalities in nutritional status,
and altered consciousness are the most consistently reported
risk factors for pressure ulcers. Pain, infectious complications,
prolonged and expensive hospitalizations, persistent open ul-
cers, and increased risk of death are all associated with the
development of pressure ulcers. A pressure ulcer can increase
a patient’s length of stay (LOS) in hospital, pain, and infection,
as well as contributing to mortality [11, 12]. According to the
National Pressure Ulcer Advisory Panel (NPUAP), the inci-
dence range may vary from 0.4 to 38 % in hospitals [13].
Normally, skin and mucus membranes of human beings
harbor diverse groups of microorganisms that can be
widely divided into two major groups including resident
flora, which consists of comparatively fixed types of mi-
croorganisms. The transient flora consists of nonpatho-
genic or potentially pathogenic microorganisms that in-
habit the skin or mucous membranes for hours to weeks.
Bessa [14] reported the most common bacterial species to
b e S t a p h y l o c o c c u s a u re u s 3 7 % , f o l l o w e d b y
Pseudomonas aeruginosa 17 %, Proteus mirabilis 10 %,
Escherichia coli 6 %, and Corynebacterium spp. 5 %.
Polymicrobial infection was found in 27.1 % of the sam-
ples [14]. Most of the acute and chronic wound infections
contained consortia of both aerobic and anaerobic micro-
biota. The predominant skin resident microbiota are aero-
bic and anaerobic, such as diphtheroid bacilli (e.g.,
Corynebacterium, Propionibacterium), non-hemolytic
aerobic and anaerobic staphylococci (S. epidermidis and
other coagulase-negative staphylococci, occasionally
S. aureus, and Peptostreptococcus species), Gram-posi-
tive, aerobic, spore-forming bacilli that are ubiquitous in
air, water, and soil, alpha-hemolytic Streptococci (virid-
ians streptococci) and Enterococci, and Gram-negative
coliform bacilli and Acinetobacter. Anaerobic and aerobic
bacteria often form synergistic effects to cause infections,
e.g., gangrene, necrotizing fasciitis, and cellulitis specific
to the skin and soft tissues. These bacteria are frequently
part of normal microbial flora. So, a mixture of microor-
ganisms is usually involved in many skin lesions [15].
These pathogenic microorganisms produce biofilm on
acute wounds to turn into chronic infection. Biofilm con-
sists of a sessile community of multiple bacterial species
enclosed by a protective carbohydrate-rich polymeric ma-
trix that is resistant to antimicrobial and immune cell
penetration [17]. Bacteria, whether free floating or incor-
porated within a biofilm, are particularly detrimental to
wound healing, mainly when they reach the level of crit-
ical colonization. Dowd and his colleagues isolated
Staphylococcus, Peptoniphilus, Serratia, Pseudomonas,
Finegoldia, Enterobacter, and Stenotrophomonas from
biofilms of chronic wounds [18]. Hassan and colleagues
have found associated S. epidermidis, S. aureus, E. coli,
E. faecalis, P. aeruginosa, and K. pneumoniae with bio-
film formation in chronic wounds [19]. P. aeruginosa,
staphylococci species, and E. coli were found to be the
most common biofilm producers on chronic wounds.
Diverse microbial flora on wounds make the treat-
ment of patients more difficult, especially when a large
number of resistance microorganisms are found on
biofilms of chronic wounds. Resistance is increasing
because of biofilm matrix production, horizontal gene
transfer, daily use of antibiotics, and microbial mutation.
Resistance to antibiotics in biofilm-producing bacteria is
a crucial problem in the management and treatment of
chronic wounds [20, 21]. Improvement in clinical prac-
tices is possible if biofilm-producing bacteria are isolat-
ed and characterized from chronic wounds and their
antibiotic resistance patterns are determined.
Acute Wounds
Wounds are the major cause of morbidity worldwide. Many
studies have reported that for every million patients with
wounds, at least 10,000 died from microbial infection [1].
There is only the skin which works as a vital organ and serves
as a protective barrier between the external environment and
human body [2]. Breaks in the skin due to many issues, such
as ulcers or traumatic wounds, expose the subcutaneous tis-
sue, providing suitable moisture, nutritive conditions, and
temperature for microbial colonization [3]. Wound healing is
a highly active process including coagulation, inflammation,
cell proliferation, and tissue remodeling, which leads to rapid
closure of the wound within 3–14 days [22]. The symptoms of
acute wounds are localized bleeding, pain, redness, swelling,
and pus production at the site of wounds. However, symptoms
of acute wounds may also vary according to the nature and
type of wound. For management of such wounds, emergency
techniques are employed, which usually resuscitate and re-
store optimized blood supply to the affected site in order to
control pain. If failure in pain control affects mobility, the
supply of oxygen consequently induces chronicity.
Chronic Wounds
A non-healing or chronic wound is a type of wound that does
not improve after 4 weeks or does not heal in 8 weeks [23].
Chronic wounds remain in an inflammatory stage and do not
show progression due to some specific reasons, such as the
age of the patient, in which inflammatory and physiological
response are continuously decreasing, having a major impact
on blood circulation, decrease in collagen production, and
membrane basement degeneration [24]. Similarly, other rea-
sons are also responsible for not allowing the wounds to heal
within the proper time frame, in which the most important is

Fig. 2 Types of chronic wounds observed in studied patients. a Venous leg ulcer, b pressure ulcer, c abscess ulcer, d surgical ulcer, and e others
bacterial toxin production, causing collagen degradation,
stress, and malnutrition, preventing the normal healing of
wounds [25]. Kane [26] also reported that stress delays the
process of wound healing (Fig. 3).
Chronic wounds are affecting approximately 25 % of indi-
viduals with diabetes [27]. Unfortunately, the result of DFU
can be amputation in some patients. Bakker and Riley [28]
have reported that globally, a patient with DFU loses a lower
limb every 30 s [29]. Foot ulceration in diabetic patients is
significantly associated with mortality and morbidity. The
mortality rate is 39–80 % in patients after development of
diabetic foot ulcer [30]. Moreover, DFU is a leading cause
of morbidity and hospitalization. Usually, patients stay for
long time in hospital as compared to other complications de-
veloped by diabetes patients [7].
Globally, the incidence rate of DFU is 1–4 % [31] and
prevalence rate is 5.3–10.5 % [30]. In a population-based
study, it has been reported that DFU causes 75,000 deaths
annually in the UK [32]. In the USA, DFU is the sixth main
cause of death [33], and 8 % prevalence of DFU is reported in
the USA [34]. The International Diabetes Federation reported
16.8 % prevalence rate of DFU in Saudi Arabia [35]. In a
study on randomly selected patients with diabetes in
Sweden, an incidence of 3.6 % has been reported, and another
study in the Netherlands found an incidence rate of 2.1 % in
patients with type 2 diabetes. Foot ulcer in Ethopian diabetics
results in 12 % of patients dying [36]. In an epidemiological
study, Pendsey and colleagues found 3 % prevalence of DFU
in younger Indian diabetic patients [37]. Studies from Pakistan
showed 4 and 10 % prevalence of foot ulceration in diabetic
patients, and the amputation rate was found within the range
of 8–21 % following DFU [38].
K. Rahim et al.
It is believed that vascular disease, ischemia, neuropathy,
foot traumas, Charcot’s joint, and foot deformities may lead to
DFU [39–41]. A hospital-based study in Ethiopian diabetic
patients identified poor follow-up and reduced glycosemic
control as a potential factor in development of DFU [42]. In
a cross-sectional study, overweight and poor self-care practice
were recorded as contributing factors in the development of
DFU in diabetic patients in rural residences [43]. In another
study from Saudi Arabia, Ahmad and his colleagues observed
that diabetic patients with inappropriate footwear, burns, ex-
ternal injury, and tinea pedis developed foot ulcers [44].
Population-based studies from India have reported that factors
contributing to DFU are increasing age, severity of infection at
clinical presentation, low socioeconomic conditions, barefoot
walking, inappropriate footwear, and poor care of diabetes in
basic healthcare units with delayed referral of patients to spe-
cialists in secondary or tertiary healthcare units [45, 46].
Ulceration of a lower extremity is a devastating fact that not
only affects the patient directly but is also significantly asso-
ciated with economic losses since many resources are used in
treatment and prevention to reduce the progress of disease.
Venous ulcer is the predominant type of leg ulcer, accounting
for 80 % of all kinds of ulcerations [47]. It has been estimated
that about 60–80 % of leg ulcers have a venous component
[48]. Venous ulcers are caused due to the incompetency of the
venous valve and insufficiency of calf muscle pumps that
result in venous hypertension and venostasis. Consequently,
localized tissue ischemic damages are observed [49].
Simon and colleagues identified contributing risk factors
such as an artery disease, immobility, vasculitis, trauma, ear-
lier leg injuries, neoplasia, phlebitis, deep venous thrombosis,
edema, obesity, and diabetes in the development of VLU [50,
Bacterial Contribution in Chronicity of Wounds
51]. It has also been reported that the incidence of venous
ulcer increases with increasing age, obesity, diabetes, and
smoking habits [52]. In another study, chronic leg ulcer was
reported within a range of 0.6–3 % in individuals aged more
than 60 years and 5 % in individuals older than 80 years. In the
USA, venous leg ulcers were found more prevalent in older
persons, particularly women, and accounted for 1 % [53].
Margolis and colleagues also found higher prevalence of
VLU in women [54]. A multicenter study from Pakistan found
34.8 % prevalence of VLU in patients with history of deep
vein thrombosis, lack of exercise, and family history of chron-
ic venous disease [55].
VLU is a leading cause of morbidity, and the prevalence
rate ranges between 1.9 and 13.1 % in the community [56]. A
study by Sasanka reported that approximately 10 % of indi-
viduals may develop chronic leg ulcer during the course of
their life, with mortality rate of 2.5 % [57]. The incidence of
chronic leg ulcers in patients hospitalized at surgical units was
found to be between 1.5 and 20.3 % in China [58]. An epide-
miological study conducted on chronic wounds in India esti-
mated the prevalence of venous leg ulcer to be 4.5 per 1000 in
the population [59]. In a population-based study conducted in
Australia, it was found that leg ulcers affect 1.1 per 1000 [60].
A study from Brazil reported prevalence of 35.5 % for vari-
cose veins and 1.5 % for chronic venous in patients with leg
ulcers [61].
Pressure ulcers (PU) are damage to the skin and underlying
tissue, caused by too much constant pressure on the skin for a
long time. They are a major health problem in individuals that
live in institutional settings [62]. Risk factors for PU include
old age, incipient dementia, physical disabilities, and comor-
bid conditions like malnutrition, edema, abnormal microcir-
culation, involuntary urination, and hypoalbuminemia, which
have an effect on the integrity and healing of soft tissue [63,
64]. The first sign of a pressure ulcer is red skin that gets
progressively worse, forms a blister, and ultimately results in
an open wound. PU most frequently occurs on shoulders,
back, elbows, back of head, buttocks, hips, ankles, and heels.
Poor oxygen supply and lack of essential nutrients in these
body parts cause damage to soft tissues and as a result avas-
cular necrosis [65, 66].
The incidence rates of PU vary significantly in clinical
settings. According to the National Pressure Ulcer Advisory
Panel (NPUAP), the incidence range of PU is 0.4–38 % in
hospitals, 2.2–23.9 % in rehabilitation services, and 0–17 % in
home healthcare [13]. Pressure ulcer is significantly associat-
ed with high morbidity and mortality; approximately 70 %
patients may die due to PU in 6 months [67]. The incidence
of PU increases due to a long stay in a healthcare setting [68].
In older hospitalized patients, PU causes increased mortality,
accounting for 25–30 % [69]. A study from Sweden figured
out 28.2 % prevalence of pressure ulcers, but prevalence fell to
26.7 % in surgical units [70]. In Europe, the prevalence of PU
was found to be 18.1 % [71]. Furthermore, large variations in
the prevalence of PU have been reported in Intensive care
units (ICUs) throughout the world, accounting for 49 % in
regions of Western Europe [72], 50 % in Australia [73, 74],
22 % in North America [72], and 29 % in Jordan [75]. Results
of prevalence surveys conducted in nursing homes of the
Netherlands and Germany showed that the prevalence rate is
30.8 % in the Netherlands and 8.3 % in Germany [76].
Surgical site infection (SSI) is defined as an infection oc-
curring within 30 days after a surgical operation or within
1 year if an implant is left in place after procedure and affects
either incision or deep tissues at the operation site. These
infections may be superficial, or deep incisional infection, or
infections involving organ or body space [77]. Postoperative
surgical infection is among the most common problems for
patients who undergo operative procedures and the third most
frequently reported nosocomial infection in the hospital pop-
ulation [77]. Postoperative surgical site infections are associ-
ated with increased morbidity, mortality, prolonged hospital
stay, and increased economic costs for patient care [78]. It has
also been reported that these surgical site infections remain
common causes of morbidity and mortality due to emergence
of antimicrobial-resistant pathogenic bacteria [77, 78].
Surgical site infections can be reduced by appropriate use
of surgical antimicrobial prophylaxis. In hospital practice, 30–
50 % of antibiotics are prescribed for surgical prophylaxis and
30–90 % of this prophylaxis is inappropriate [79]. This inap-
propriate use increases selection pressure, favoring emergence
of drug-resistant pathogenic bacteria [80], which makes the
choice of empirical antimicrobial agents more difficult, and
hence increases the risk of postoperative wound infections.
Studies on the magnitude of surgical infections are complicat-
ed by the heterogeneous nature of these infections. The prev-
alence and incidence rates of postoperative wound infections
vary widely between procedures, hospitals, surgeons, patients,
and geographical locations [81, 82]. A study has documented
a high rate of SSI in dirty surgery (60 %), compared with
contaminated (27.3 %), clean contaminated (19.3 %), and
clean surgery (14.3 %), the association being statistically sig-
nificant [81]. Studies have shown that introduction of minimal
invasive surgery, like laparoscopic surgery, has resulted in a
decrease in the incidence of SSI. A review by Boni [83] re-
ported a decrease in SSI in patients with acute appendicitis to
2 % with minimally invasive procedures compared to 8 %
with open procedures. Similarly, in patients undergoing cho-
lecystectomy, the incidence of SSI was reported to be low
(1.1 %) with laparoscopy in comparison with 4 % following
open surgery [82].
Several literature sources report varying SSI prevalence
and incidence rates in different parts of the world, ranging
from 5.6 to 26 % [84]. A study at a University Hospital in
Brazil among general surgical patients reported a high rate,
reaching 16.9 % of these infections, with a higher rate in clean

contaminated (17.8 %) as compared to contaminated surgery
(12.5 %). Nevertheless, most interventions involved the diges-
tive tract [84]. But, a study at [83] a hospital in Vietnam re-
ported a high incidence rate, 15.2 %, of surgical infections
among orthopedic patients, which is higher than that of
France, with no difference in the surgical infection rate be-
tween patients who received appropriate antimicrobial pro-
phylaxis and patients who did not (4.4 vs. 6.4 %, P = 0.4)
[85]. A study in Kampala, Uganda, among surgical patients,
reported 9.64 % of surgical site infections in the pre-
intervention phase, in which no antimicrobial prophylaxis
was administered, as compared to 2.56 % in the intervention
phase in which antimicrobial prophylaxis was administered
preoperatively [86]. Limited studies have been done in
Pakistan to determine the extent to which surgical site infec-
tions occur. In one of those few studies, a prevalence rate of
9.294 % of surgical site infections was reported among hos-
pitalized surgical patients [87].
In most postoperative SSIs, the causative pathogens origi-
nate from endogenous flora of the patient’s skin, mucous
membranes, or hollow viscera [77]. The most commonly iso-
lated bacterial pathogens are Staphylococcus aureus,
Enterobacteriaceae, Coagulase Negative Staphylococci,
Enterococci, and Pseudomonas aeruginosa [88, 89].
Although the pathogens isolated depend on the surgical pro-
cedure involved, recent reports have documented an increas-
ing proportion of Gram-positive organisms and a decrease in
the number of Gram-negative organisms associated with SSIs
[77, 85]. Furthermore, there is an increase in incidence of SSIs
attributed to antimicrobial-resistant pathogenic bacteria like
methicillin-resistant S. aureus (MRSA) [90, 91] and
vancomycin-resistant S. aureus.
A recent study in the USA among patients who underwent
operation for hollow viscus injury documented Escherichia
coli as the most commonly isolated microorganism (64.7 %)
followed by Enterococci species (41.2 %) and Bacteroides
(29.4 %) [92]. Another recent study at a University hospital
in Iran reported S. aureus to be the commonest bacteria path-
ogen (43 %), followed by E. coli (21 %), Klebsiella spp.
(13 %), Pseudomonas (10 %), and CoNS (5 %) among surgi-
cal patients [93]. In that study, MRSA accounted for a rate of
78.9 % of all S. aureus isolates [93]. Another study reported
that MRSA was the most frequent pathogen recovered and the
prevalence rate of MRSA SSI almost doubled during the study
period, increasing from 0.12 infections per 100 procedures to
0.23 infections per 100 procedures [91].
Bacterial contamination of the surgical site is a prerequisite
for SSIs. The sensitivity pattern of SSI isolates is changing
due to increasing emergence of antimicrobial-resistant biofilm
producing pathogenic bacteria strains like MRSA [91], making
the choice of empirical treatment more difficult and expen-
sive. The magnitude of antimicrobial resistance among
biofilm-producing bacteria globally is unknown, and this is
K. Rahim et al.
particularly the case in developing countries where few data
are available [94].
Bacterial Flora of Chronic Wounds
Chronic wounds are colonized by diverse populations of bacte-
ria [95, 96], which directly and indirectly contribute to chronic
wound phenotypes [97]. Colonizing bacteria such as
Staphylococcus epidermidis and Corynebacterium species live
on the skin and usually prevent colonization of pathogenic bac-
teria [98]. However, colonization of pathogenic bacteria at
wound bed is associated with wound chronicity. Most chronic
wounds contain more than one bacterial species and produce a
synergetic effect that results in previously non-virulent bacterial
species becoming virulent and causing damage to the host [99].
According to Wolcott [100], chronic wounds contain a diverse
microbiota such as Staphylococcus aureus, Staphylococcus
epidermidis, Pseudomonas aeruginosa, Stenotrophomonas
maltophilia, Finegoldia magna, Enterococcus faecalis,
Corynebacterium striatum, Staphylococcus haemolyticus,
Propionibacterium acnes, Corynebacterium
tuberculostearicum, Anaerococcus vaginalis, Staphylococcus
lugdunensis, Delftia acidovorans, Streptococcus agalactiae,
Acinetobacter baumannii, Proteus mirabilis, Streptococcus
salivarius, Serratia nematodiphila, Ralstonia pickettii,
Fusobacterium nucleatum, Staphylococcus pettenkoferi,
Staphylococcus lugdunensis, Enterobacter hormaechei,
Prevotella bivia, Corynebacterium jeikeium, Bacteroides
fragilis, and Flavobacterium succinicans, confirmed by 16S
rDNA pyrosequencing analyses [101]. Shin Yw in Malaysia
also reported the prevalence of microbiota on wounds
Staphylococcus aureus 23.3 % and Pseudomonas aeruginosa
14.8 % that were the most prevalent bacteria found in wounds.
Staphylococcus aureus was found significantly more often in
patients with chronic wounds (48.8 %) than in patients with
acute wounds (9.5 %), while Staphylococcus epidermidis was
found predominantly in acute wounds (17.9 %). At the time of
study, patients with chronic wounds (58.3 %) had received more
antibiotic treatments in the past previous 12 months compared
with patients with acute wounds (16.7 %) [101]. A study by
Thomas reported that individuals with chronic wounds are usu-
ally short of sufficient oxygen due to poor perfusion, and anaer-
obic bacteria take advantage of this condition, multiply, and
form microcolonies instead of aerobic bacteria [102].
Moreover, host factors like age, immunosuppression, medica-
tion, and concomitant diseases may also influence healing of
chronic wounds (https://www.betterhealth.vic.gov.
au/health/conditionsandtreatments/wounds-how-to-care-for-
them).
While investigating bacterial diversity in chronic wounds,
Dowd [18] reported Staphylococcus, Pseudomonas,
Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia,
and Serratia. Similarly, coagulase negative staphylococci and
Bacterial Contribution in Chronicity of Wounds
Fig. 3 Major healing and non-
healing differences between acute
and chronic wounds [16]
S. aureus have been isolated as the predominant bacterial spe-
cies from chronic wounds [96]. The most frequently identified
pathogen is P. aeruginosa, and this accounted for 7–33 % of
ulcers [103]. A bacterial culture study conducted by Fazli and
colleagues recognized S. aureus at the surface of wounds and
Pseudomonas aeruginosa in deeper layers of tissue [104].
A first-time study reported Bordetella trematum from a dia-
betic leg ulcer patient, but the causative role of this pathogen was
not clear in diabetic foot infection [105]. Other studies on patients
with diabetic foot ulcer documented MRSA as potential patho-
gens, and this was found to increase the time of wound healing
process twofold [106, 107]. Wysocki [108] isolated several bac-
terial species from patients with VLU, but only Staphylococcus
aureus and Pseudomonas aeruginosa were recognized as sole
wound pathogens. Recently, bacterial diversity was investigated
in German patients with chronic leg ulcers, and Staphylococcus
aureus was found in 50 % cases, P. aeruginosa and
Enterobacteriaceae in 30 % cases and MRSA in approximately
10 % cases [109]. Bacterial colonization of PU in Italian patients
who received care at home was investigated; MRSA,
Enterococcus, and bacilli species were identified as multidrug-
resistant species of PU [110]. A prospective cohort study includ-
ed patients with pressure ulcers and hospitalized in a basic health
care unit and identified S. aureus in 20.7 % cases and bacilli
species in 32.5 % cases [111]. These microbiota diversities and
significant variabilities cause difficulties in selecting proper anti-
biotics to cure chronic wounds.
Biofilm Formation
Naturally, attachment of microorganism on the surface
and microbial proliferation which causes many serious
problems is defined as biofilm. The biofilm is further
divided in many types of infections; here, we will define
some of the special cases like periodontitis, chronic lung
infection in cystic fibrosis, catheters infections, and the
major case of biofilm in chronic wounds. Chronic peri-
odontal infections are common, and the prevalence of
severe periodontitis in Western countries has been esti-
mated to be 5–15 % [112]. In Cystic fibrosis, bacterial
biofilm is produced inside lungs and reduced its function
which lead to death of the patient, Pseudomonas
aeruginosa biofilms are highly found in this type of in-
fection [113], and biofilm on urinary catheters results in
persistent infections that are resistant to antibiotic [114].
Recently, it has been observed the bacterial roles in
chronic wounds infection which were analyzed by light
and scanning electron microscopy [115]. Bacteria on the
wound bed multiply, form micro-colonies, and secrete
extracellular polymeric substances for formation of bio-
film matrix. Usually, components of biofilm are proteins,
polysaccharides, and neutrophil or bacterial cell extracel-
lular DNA. However, the composition of biofilm may
vary depending on type of bacterial strain and chronic
wound conditions [116, 117]. The bacterial species in
single or mixed colonies take proteinaceous substrates
and nutrients from moist wound bed that provides an
ideal environment for development of biofilm [118].
Moreover, bacterial communities within biofilm matrix
are resistant to antimicrobial agents, antibodies, and
toxins. The biofilm matrix formation consists of three
phases: initial and irreversible attachment, establishment
of bacterial microcolonies, and dispersion of mature bio-
film. In the last phase, cells from biofilm are dispersed

and migrate to other surfaces to spread biofilm, and consequently,
the transition from local to systemic infection may occur [119].
It has been estimated that about 65 % of infections caused
by microorganisms are significantly associated with formation
of biofilms, and cells in biofilm show greater resistance to
antimicrobial agents as compared to planktonic bacterial cells
[120]. Many studies have verified that several bacteria within
chronic wounds develop biofilms. Using of peptide nucleic
acid-based fluorescence in situ hybridization (PNA-FISH)
and confocal laser scanning microscopy Fazli and his team
isolated S. aureus from outer layers and P. aeruginosa from
deeper layers of infected tissue and found it associated with
biofilm formation [121]. Another study from Pakistan report-
ed that 54.78 % S. aureus strains produced biofilms in chronic
wounds [122]. However, Attinger and Wolcott found biofilm-
producing bacteria and fungi using PCR which are associated
with more than 90 % of chronic wounds [123]. Clinical iso-
lates were evaluated in a study, and different strains of
S. epidermidis, P. aeruginosa, and S. aureus were found pro-
ducing biofilms [124]. Similarly, Dowd and colleagues isolat-
ed Staphylococcus, Peptoniphilus, Serratia, Pseudomonas,
Finegoldia, Enterobacter, and Stenotrophomonas from de-
bridement of chronic wounds [18]. Hassan and colleagues
found S. epidermidis, S. aureus, E. coli, E. faecalis,
P. aeruginosa, and K. pneumoniae associated with biofilm
formation in chronic wounds [19]. Moreover, Schaber [125]
studied P. aeruginosa for biofilm formation in chronic burn
wounds and found that P. aeruginosa are quickly colonized in
burn wounds and produce biofilms around blood vessels.
Harris et al. also showed the biofilm phenotype of the bacterial
isolates that were determined using confocal laser scanning
microscopy (CLSM) and image analysis [126], as in previous
studies also proved the bacterial biofilm [127–130].
Antibiotics Resistance
Antimicrobial agents, particularly antibiotics, are considered
to suppress the growth of metabolically active bacterial cells
in biofilm, which usually have detrimental effects on the host
tissue due to their ability to initiate inflammation [131].
However, biofilm is composed of dormant cells in large pro-
portion that exhibit resistance to antibiotics [132]. Actually,
the biofilm matrix acts as a diffusion barrier either by reducing
the rate of antibiotic movement to the interior of biofilm or by
reacting chemically with the antibiotic to delay its penetration
into biofilm [133]. If the antibiotic takes a longer time to
penetrate into biofilm than the time required for antibiotic
treatment, then this slow diffusion may be a reason for ob-
served resistance to antibiotics [134]. Moreover, Lewis in
[135] reported that the diffusion of positively charged antibi-
otics, for example aminoglycosides, is prevented by negative-
ly charged biofilm matrix either by molecular binding or
chemical interaction [135].
K. Rahim et al.
In a mature biofilm, bacteria grow slowly due to the defi-
ciency of nutrients that result in resistance to antibiotics of
bacteria within biofilm. In one study, slow growth rate was
observed for biofilm producing P. aeruginosa, E. coli, and
S. epidermidis, and these were found to be resistant to cipro-
floxacin [136]. Furthermore, due to greater utilization of oxy-
gen and glucose in the outer layers, communities of anaerobic
bacteria are produced deep in the biofilm, where these cells
grow very slowly. Consequently, those antibiotics, which pen-
etrate into the biofilm, do not kill slow-growing bacterial com-
munities within the biofilm [137]. In an investigation, Dowd
[18] isolated 62 % of anaerobic biofilm-producing bacteria
from pressure ulcer.
The biofilm age is an important factor that contributes to
antibiotic resistance. Rani and colleagues observed that 10-
day-old biofilms are more resistant than 2-day-old biofilms
[138]. This fact emphasizes the need for timely diagnosis as
well as treatment. Resistance to antibiotics is greatly enhanced
by the horizontal gene transfer mechanism in which resistant
plasmids are transferred from biofilm-producing bacteria to
non-biofilm-producing bacteria. Such plasmids show resis-
tance to antibiotics such as beta-lactams, aminoglycosides,
tetracycline, erythromycin, sulfonamides, trimethoprim, and
glycopeptides. Extensive bacterial communities and, conse-
quently, a greater chance of contact between these cells in
the biofilm matrix significantly favor the horizontal transfer
of resistant plasmid. Hennequin [139] reported that
K. pneumonia is the most prominent bacteria due to its ability
to transfer resistant plasmid at the rate of approximately 0.5/
donor cells.
Most bacterial cells present in outer layers of the bio-
film frequently come in contact with antibiotics and are
more susceptible to various antibiotics. These exposed
cells in the biofilm quickly die on exposure to antibiotics,
for example, ciprofloxacin. However, it is believed that
the persistent cells in the biofilm are found to be resistant
to the entire removal of biofilm-producing bacteria. These
wild-type persistent cells remain in a dormant state in the
presence of antibiotics and usually tolerate them [140].
Roberts and Steward found about 0.1 to 10 % persistent
cells in a biofilm [141]. Furthermore, Lewis described that
when antibiotic treatment is stopped, the persistent cells
become metabolically active to restart the formation of
biofilm [16]. In another study, the antibiotic susceptibility
of biofilm-producing E. coli was investigated, and three
different antibiotics including ampicillin, ofloxacin, and
kanamycin were applied to bacterial cells deep in layers
of mature biofilms. The results of this study revealed that
kanamycin and ofloxacin were found to be effective anti-
biotics against biofilm cells, while ampicillin was found
to be less effective against these cells; consequently, bio-
film formation again started when the ampicillin therapy
was stopped [142].
Bacterial Contribution in Chronicity of Wounds
Prevalence of diabetes and venous disease is significantly
high in the Pakistani population due to lack of basic health
care facilities and late referral of patients to tertiary care hos-
pitals [55]. According to Wound Healing Association of
Southern Africa (WHASA), biofilm formation is the most
important virulence factor that leads to chronic implications
such as foot, leg, and pressure ulcers [143]. Resistance of
biofilm-producing bacteria to antibiotics is a crucial problem
in the management and treatment of chronic wounds [21].
Improvement in clinical practices is possible if biofilm-
producing bacteria are isolated and characterized from chronic
wounds and their antibiotic resistance patterns are determined.
Conclusion
Chronic wounds are found more prevalent in every case of
wounds. The main cause of a chronic wound is direct micro-
bial contact with the wound and its proliferation on it. Further,
microbes become involved in biofilm production, which
shows high resistance toward healing and antibiotic action.
During biofilm production, most of the normal bacteria be-
come pathogenic, due to horizontal gene transfer from patho-
genic bacteria. Most aerobic bacteria become involved in non-
healing of wounds because of direct environmental contact.
This review concludes that acute wounds become chronic
because of non-hygienic environmental conditions, presenting
a sever threat to human beings in developing countries.
Acknowledgments This work was granted by CAPES, CNPq,
FUNDECT, and FAPDF.
References
1. Bygott JD, Bertram BCR, Hanby JP (1979) Male lions in large
coalitions gain reproductive advantages. Nature 282:839–841
2. Fallouh HB, Venugopal PS, Newton A (2009) Occult gunshot
wounds in the emergency department. Lancet 373:631–632
3. Dombradi Z, Bodnar F, Orosi P, Dombrádi V, Szabo J (2009) A
case report of vancomycin-resistant Enterococcus faecalis coloni-
zation of a femoral wound in central Europe. Cent Eur J Med 4:
259–261
4. Nasser S, Mabrouk A, Maher A (2003) Colonization of burn
wounds in Ain Shams University Burn Unit. J Int Soc Burn Inj
29:229–233
5. Quinn JV (1998) Tissue adhesives in wound care. BC Decker
6. Reiber GE, Ledoux WR (2002) Epidemiology of diabetic foot
ulcers and amputations: evidence for prevention. Evid Base
Diabetes Care 641–665
7. Ramachandran A (2004) Specific problems of the diabetic foot in
developing countries. Diabetes Metab Res Rev 20:123–133
8. Gloviczki P, Gloviczki ML (2009) Evidence on efficacy of treat-
ments of venous ulcers and on prevention of ulcer recurrence.
Perspect Vasc Surg Endovasc Ther 21:259–268
9. Bergan JJ, Schmid-Schönbein GW, Smith PD, Nicolaides AN,
Boisseau MR, Eklof B (2006) Chronic venous disease. N Engl J
Med 355:488–498
10. Smyth ET, McIlvenny G, Enstone JE, Emmerson AM,
Humphreys H, Fitzpatrick F, Davies E, Newcombe RG, Spencer
RC (2008) Hospital Infection Society Prevalence Survey Steering
Group. Four country healthcare associated infection prevalence
survey 2006: overview of the results. J Hosp Infect 69:230–48
11. Armour-Burton T, Fields W, Outlaw L, Deleon E (2013) The
healthy skin project: changing nursing practice to prevent and treat
hospital-acquired pressure ulcers. Crit Care Nurse 33:32–40
12. Chicano S, Drolshagen C (2009) Wound care: reducing hospital-
acquired pressure ulcers. J Wound Ostomy Cont Nurs 36:45–50
13. Cuddigan J, Berlowitz DR, Ayello EA (2001) Pressure ulcers in
America: prevalence, incidence, and implications for the future.
Adv Skin Wound Care 14:208
14. Bessa LJ, Fazii P, Giulio M, Cellini L (2015) Bacterial isolates
from infected wounds and their antibiotic susceptibility pattern:
some remarks about wound infection. Int Wound J 12:47–52
15. Olszewski WL (2016) Role of bacteria in pathogenesis of lower
leg ulcers. In ulcers of the lower extremity 125–140. Springer,
India
16. Lewis K (2012) Persister cells: molecular mechanisms related to
antibiotic tolerance. In Antibiotic resistance 121–133. Springer
Berlin Heidelberg
17. Ghannoum M, O’Toole GA (2008) Microbial biofi lms. ASM
Press, Washington, DC
18. Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA,
Wolcott RD (2008) Survey of bacterial diversity in chronic
wounds using pyrosequencing, DGGE, and full ribosome shotgun
sequencing. BMC Microbiol 8:1
19. Hassan M, Kjos M, Nes IF, Diep DB, Lotfipour F (2012) Natural
antimicrobial peptides from bacteria: characteristics and potential
applications to fight against antibiotic resistance. J App Microbiol
23:1365–2672
20. Mah MW, Memish ZA, Cummingam G, Bannyamm RM (2001)
Outbreak of acenetobacter baumanii in intensice care unit associ-
ated with trechostaumi. Am J Infect Control 29:284–288
21. Stewart PS, Costerton JW (2001) Antibiotic resistance of bacteria
in biofilms. Lancet 358:135–138
22. Davis SC, Ricotti C, Cazzaniga A (2008) Microscopic and phys-
iologic evidence for biofilm-associated wound colonization
in vivo. Wound Repair Regen 16:23–29
23. Mustoe TA, Shaughnessy KO, Kloeters O (2006) Chronic wound
pathogenesis and current treatment strategies: a unifying hypoth-
esis. Plast Reconstr Surg 117:35–41
24. Doughty MJ, Laiquzzaman M, Muller A, Oblak E, Button NF
(2007) Central corneal thickness in European (white) individuals,
especially children and the elderly, and assessment of its possible
importance in clinical measures of intra-ocular pressure.
Ophthalmic Physiol Opt 22:491–504
25. Evans E (2005) Nutritional assessment in chronic wound care. J
Wound Ostomy Cont Nurs 32:317–320
26. Kane DP (2007) Chronic wound healing and chronic wound man-
agement. J Wound Regen 78:123–138
27. Singh N, Armstrong DG (2005) Preventing foot ulcers in patients
with diabetes. J Acquir Manag 293:217–228
28. Bakker K, Riley PH (2005) The year of the diabetic foot. Diabetes
Voice 50:11–14
29. Bahler K, Riley PH (2005) The year of the diabetic foot. Diabetes
Voice 51:15–18
30. Reiber GE (2001) Epidemiology of foot ulcers and amputations in
the diabetic foot. Diabet Foot 9:13–19
31. Reiber GE, Ledoux WR (2002) Epidemiology of diabetic foot
ulcers and amputations: evidence for prevention. Evide Base
Diabetes Care Wiley 641–665

32. McInnes AD (2012) Diabetic foot disease in the United Kingdom:
about time to put feet first. J Foot Ankle Res 5:1146–1186
33. Kung HS, Hoyer DL, Xu J (2005) Deaths: final data for national
vital statistics. Braz J Microbiol 58:2004–2041
34. Margolis DJ, Malay DS, Hoffstad OJ, Leonard CE, MaCurdy T,
Tan Y, Molina T, de Nava KL, Siegel KL (2011) Economic burden
of diabetic foot ulcers and amputations
35. International Diabetes Federation (2014) Diabetes Atlas
http://www.eatlas.idf.org/media
36. Pittet D, Harbarth S, Sudan R (2011) Andreas Voss International
Conference on Prevention & Infection Control (ICPIC 2011).
BMC Proc 5
37. Pendsey S, Abbas ZG (2007) The step-by-step program for reduc-
ing diabetic foot problems: a model for the developing world. Curr
Diab Rep 7:425–428
38. Ali SM, Basit A, Shaikh T, Mumtaz S, Hydrie MZ (2001) Diabetic
foot ulcer-a prospective study. J Pak Med Assoc 51:78–81
39. Reiber GE, Vileikyte L (1999) Causal pathways for incident
lower-extremity ulcers in patients with diabetes from two settings.
Diabetes Care 22:157–176
40. Wieman TJ (2003) Principles of management: the diabetic foot.
Am J Surg 9:190–295
41. Marston WA (2003) Infection in daibetic foot ulcer. Diabetes Care
26:1701–1705
42. Brem H, Sheehan P, Rosenberg HJ, Schneider JS, Boulton AJ
(2006) Evidence-based protocol for diabetic foot ulcers. Plast
Reconstr Surg 117:193–209
43. Deribe B, Woldemichael K, Nemera G (2014) Prevalence and
factors influencing diabetic foot ulcer among diabetic patients at-
tending Arbaminch Hospital South Ethiopia. J Diabetes Metab 5:
4172–6156
44. Ahmad W, Khan IA, Salma G, Farhan KAS, Khan I (2013) Risk
factors for diabetic foot ulcer. J Ayub Med Coll Abbottabad 25:1–2
45. Rathur HM, Boulton AJ (2007) The diabetic foot. Clin Dermatol
25:109–120
46. Viswanathan V (2007) The diabetic foot: perspectives from
Chennai, South India. Int J Extrem Wounds 6:34–6
47. Spentzouris G, Labropoulos N (2009) The evaluation of lower-
extremity ulcers seminar. Int Radiol 26:286–295
48. Baker R, Fraser RC (1995) Development of review criteria:
linking guidelines and assessment of quality. Bio Med J 311:
370–373
49. Mark H (2005) Lower extremity venous anatomy. Semin Interv
Radiol 22:147–156
50. Nelson EA, Bell-Syer SE, Cullum NA, Webster J (2000)
Compression for preventing recurrence of venous ulcers.
Cochrane Database Syst Rev 4:CD002303
51. Simon LS, Mills JA (2009) Nonsteroidal anti-inflammatory drugs.
New Eng J Med 302:1179–1185
52. Agale SV (2013) Chronic leg ulcers: epidemiology,
aetiopathogenesis, and management. Ulcers
53. Abbade LP, Lastoria S, Almeida HR, Stolf HO (2005) A
sociodemographic, clinical study of patients with venous ulcer.
Int J Dermatol 44:989–992
54. Margolis DJ, Bilker W, Santanna J, Baumgarten M (2002) Venous
leg ulcer: incidence and prevalence in the elderly. J Am Acad
Dermatol 46:381–392
55. Khan AF, Chaudhri R, Ashraf MA, Mazaffar MS, Zawar-ul-Imam S,
Tanveer M (2013) Prevalence and presentation of chronic venous
disease in Pakistan: a multicentre study. Phlebology 28:74–79
56. Rayner RCK, Keaton J, Prentice J, Santamaria N (2009) Leg ul-
cers: atypical presentations and associated comorbidities. Wound
Pract Res 17:168–185
57. Sasanka CS (2012) Venous ulcers of the lower limb: where do we
stand. Ind J Plast Surg 45:266–274
K. Rahim et al.
58. Fu X (1998) Skin ulcers in lower extremities: the epidemiology
and management in China. Int J Low r Extrem Wounds 4:4–6
59. Shukla A, Rasik AM, Patnaik GK (2005) Depletion of reduced
glutathione, ascorbic acid, vitamin E and antioxidant defence en-
zymes in a healing cutaneous wound. Free Radic Res 26:93–101
60. Baker SE, Hopkinson SB, Fitchmun M, Andreason GL, Frasier F,
Plopper G, Quaranta V, Jones JC (1996) Laminin-5 and
hemidesmosomes: role of the alpha 3 chain subunit in
hemidesmosome stability and assembly. J Cell Sci 109:2509–
2520
61. Faria E, Blanes L, Hochman B, Filho MM, Ferreira L (2011)
Health-related quality of life, self-esteem, and functional status
of patients with leg ulcers. Wounds UK 23:4–10
62. Lyder CH (2003) Pressure ulcer prevention and management.
JAMA 289:223–226
63. Fogerty MD, Abumrad NN, Nanney L, Arbogast PG, Poulose B,
Barbul A (2008) Risk factors for pressure ulcers in acute care
hospitals. Wound Repair Regen 16:11–18
64. Lyder C, Ayello E (2008) Pressure ulcers: a patient safety issue.
Agency Healthcare Res Qual 8:1–33
65. Bluestien D, Jawaheri A (2008) Pressure ulcers: prevention, eval-
uation, and management. Am Fam Physician 78:1186–1194
66. European Pressure Ulcer Advisory Panel (2009) Prevention and
treatment of pressure ulcers. http://www.npuap.org/wp-
content/uploads/2012/03/Final-2009-Treatment-Technical-
Report1
67. Brown G (2003) Long-term outcomes of full-thickness pressure
ulcers: healing and mortality. Ostomy Wound Manage 49:42–50
68. Horn SD, Bender SA, Ferguson ML, Smout RJ, Bergstrom N,
Taler G, Cook AS, Sharkey SS, Voss AC (2004) The national
pressure ulcer long‐term care study: pressure ulcer development
in long‐term care residents. J Am Geriatr Soc 52:359–367
69. Grey JE, Harding KG, Enoch S (2006) Pressure ulcers. Braz Med
J 332:472–475
70. Gunningberg L, Lindholm C, Carlsson M, Sjoden PO (2000)
Effect of visco-elastic foam mattresses on the development of
pressure ulcers in patients with hip fractures. J Wound Care 9:
455–460
71. Vanderwee K, Clark M, Dealey C, Gunningberg L, Defloor T
(2007) Pressure ulcer prevalence in Europe: a pilot study. J Eval
Clin Pract 13:227–235
72. Shahin S, Jafari A, Mobli H, Rafiee S, Karimi M (2008) Effect of
farm size on energy ratio for wheat production: a case study from
Ardabil province of Iran American. Eurasian J Agric Environ Sci
3:604–608
73. Ballard N (2008) How our ICU decreased the rate of hospital
acquired pressure ulcers. J Nurs Care Qual 23:92–96
74. Elliot R, Mckinley S, Fox V (2008) Quality improvement program
to reduce the prevelence of pressure ulcer in intensive care unit
(ICU). Am J Crit Care 17:328–324
75. Tubaishata A, Anthonyb D, Salehc M (2011) Pressure ulcers in
Jordan: a point prevalence study. J Tissue Viability 20:14–19
76. Tannen A, Dassen T, Halfens R (2009) Differences in prevalence
of pressure ulcers between the Netherlands and Germany associ-
ations between risk, prevention and occurrence of pressure ulcers
in hospitals and nursing homes. J Clin Nurs 18:304–305
77. Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR
(1999) Guideline for prevention of surgical site infection. Am J
Infect Control 27:97–132
78. Weigelt JA, Lipsky BA, Tabak YP, Derby KG, Kim M, Gupta V
(2010) Surgical site infections: causative pathogens and associated
outcomes. Am J Infect Control 38:112–120
79. Graham JC, Pedler SJ (2003) Surgical antibiotic prophylaxis, 3rd
edn. Walker R and Edwards C, London, pp 569–581
Bacterial Contribution in Chronicity of Wounds
80. John WD, Salmaan K, Stephen WT (2002) Antimicrobial prophy-
laxis in surgery, 5th edn. McGraw Hill Publications, New York, pp
2111–2122
81. Ameh EA, Mshelbwala PM, Nasir AA, Lukong CS, Jabo BA,
Anumah MA, Nmadu PT (2009) Surgical site infection in chil-
dren: prospective analysis of the burden and risk factors in a sub-
Saharan African setting. Surg Infect (Larchmt) 10:105–109
82. Boni L, Benevento A, Rovera F, Dionigi G, Giuseppe MD,
Bertoglio C, Dionigi R (2006) Infective complications in laparo-
scopic surgery. Surg Infect (Larchmt) 7:101–109
83. Gastmeier P, Sohr D, Rath A, Forster DH, Wischnewski N, Lacour
M, Daschnerand F (2000) Repeated prevalence investigations on
nosocomial infections for continuous surveillance. J Hosp Infect
45:47–53
84. Santos KR, Fonseca L, Bravo NGP, Filho GPP (1997) Surgical
site infection: rates, etiology and resistance patterns to antimicro-
bials among strains isolated at Rio de Janeiro University Hospital.
Infection 25:217–220
85. Sohn AH, Tien NP, Mai VT, Van Nho V, Hanh TN, Ewald B,
Dibley M (2006) Microbiology of surgical site infections and as-
sociated antimicrobial use among Vietnamese orthopedic and neu-
rosurgical patients. Infect Control 27:855–862
86. Tiberi S, Nsubuga S, Muzaale C, Lazzarin A, Scarpellini P (2010)
Impact of preoperative antibiotic prophylaxis on the incidence of
surgical site infection in a Ugandan Hospital. Int Conference on
Healthcare Associated Infections, Fifth Decennial
87. Khan M, Khalil J, Rooh-ul-Muqim MZ, Hassan TU, Ahmed N,
Salman M, Muhammad G (2011) Rate and risk factors for surgical
site infection at a tertiary care facility in Peshawar, Pakistan. J
Ayub Med Coll Abbottabad 23
88. Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR
(1999) Guideline for prevention of surgical site infection. Am J
Infect Control 27:97–134
89. Mayhall CG (1993) Surgical infections including burns.
Prevention and control of nosocomial infections, 2nd edn.
Williams & Wilkins, Baltimore, pp 614–624
90. Weiss CA, Statz CL, Dahms RA, Remucal MJ, Dunn DL,
Beilman GJ (1999) Six years of surgical wound infection surveil-
lance at a tertiary care center: review of the microbiologic and
epidemiological aspects of 20,007 wounds. Arch Surg 134:
1041–1048
91. Haycock C, Laser C, Keuth J, Montefour K, Wilson M, Austin K,
Coulen C, Boyle D (2005) Implementing evidence‐based practice
findings to decrease postoperative sternal wound infections fol-
lowing open heart surgery. J Cardiovasc Nurs 20:299–305
92. Dellinger EP, Hausmann SM, Bratzler DW, Johnson RM, Daniel
DM, Bunt KM, Baumgardner GA, Sugarman JR (2005) Hospitals
collaborate to decrease surgical site infections. Am J Surg 190:9–15
93. Tanner J, Woodings D, Moncaster K (2006) Preoperative hair
removal to reduce surgical site infection. Cochrane Database
Syst Rev 3:1465–1478
94. Amábile-Cuevas CF, Byarugaba DK, Hsueh PR, Kariuki S,
Okeke IN (2010) Antimicrobial resistance in developing coun-
tries. Springer, New York
95. Bowler PG, Duerden BI, Armstrong DG (2001) Wound microbi-
ology and associated approaches to wound management. Clin
Microbiol Rev 14:244–269
96. Howell-Jones MJ, Wilson KEH (2005) A review of the microbi-
ology, antibiotic usage and resistance in chronic skin wounds. J
Antimicrob Chemother 55:143–149
97. Stephens P (2001) Skin and oral fibroblasts exhibit phenotypic
differences in extracellular matrix reorganization and matrix me-
talloproteinase activity. Br J Dermatol 144:229–237
98. Madsen SM, Westh H, Danielsen L, Rosdahl VT (1996) Bacterial
colonization and healing of venous leg ulcers. Apmis 104:895–
899
99. Dow G, Browne A, Sibbald RG (1999) Infection in chronic
wounds: controversies in diagnosis and treatment. Ostomy
Wound Manag 45:23–24
100. Wolcott RD, Dowd SE, Sun Y, McKeehan T, Smith E, Rhoads D
(2008) Polymicrobial nature of chronic diabetic foot ulcer biofilm
infections determined using bacterial tag encoded FLX amplicon
pyrosequencing (bTEFAP). PLoS One 3:3326
101. Shin YW, Rishya M, Sekaran M (2015) Prevalence and antibiotic
susceptibility of bacteria from acute and chronic wounds in
Malaysian subjects. J Infect Dev Ctries 9:936–944
102. Thomas S (2008) Hydrocolloid dressings in the management of
acute wounds: a review of the literature. Int Wound J 5:602–613
103. Schmidt K, Debus ES, Jessberger ST, Ziegler U, Thiede A (2000)
Bacterial population of chronic crural ulcers: is there a difference
between the diabetic, the venous, and the arterial ulcer. J Microbiol
29:62–70
104. Fazli M, Bjarnsholt T, Kirketerp-Møller K, Jørgensen B, Andersen
AS, Krogfelt KA, Givskov M, Tolker-Nielsen T (2009)
Nonrandom distribution of Pseudomonas aeruginosa and
Staphylococcus aureus in chronic wounds. J Clin Microbiol 47:
4084–4089
105. Daxboeck F, Goerzer E, Apfalter (2004) Isolation of Bordetella
trematum from a diabetic leg ulcer. Diabet Med 21:1247–8
106. Bowling FL, Stickings DS, Edwards-Jones V, Armstrong DG,
Boulton AJ (2009) Hydrodebridement of wounds: effectiveness
in reducing wound bacterial contamination and potential for air
bacterial contamination. J Foot Ankle Res 2:1–8
107. Ahmad M, Khan TH, Ansari MN, Ahmad SF (2014) Enhanced
wound healing by topical administration of d-limonene in alloxan
induced diabetic mice through reduction of pro-inflammatory
markers and chemokine expression. Biomed Central Genomics
15:10–29
108. Wysocki M, Delatour F, Faurisson F (2013) Continuous versus
intermittent infusion of vancomycin in severe staphylococcal in-
fections: prospective multicenter randomized study. Antimicrob
Agents Chemother 45:2460–2467
109. Doerler M, Eming S, Dissemond J, Wolter A, Stoffels-Weindorf
M, Reich-Schupke S, Altmeyer P, Stücker M (2014) A novel
epidermal growth factor–containing wound dressing for the treat-
ment of hard-to-heal venous leg ulcers. Adv Skin Wound Care 27:
456–460
110. Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H
(2000) Simple and reliable multiplex PCR assay for surveillance
isolates of vancomycin-resistant enterococci. J Clin Microbiol 38:
3092–3095
111. Braga L, Renner JB, Schwartz TA, Woodard J, Helmick CG,
Hochberg MC, Jordan JM (2009) Differences in radiographic fea-
tures of knee osteoarthritis in African-Americans and Caucasians:
the Johnston County Osteoarthritis Project. Osteoarthritis
Cartilage 17:1554–1561
112. Paju S, Scannapieco F (2007) Oral biofilms, periodontitis, and
pulmonary infections. Oral Dis 13:508–512
113. Niels H, Oana C, Bjarnsholt T (2010) Pseudomonas aeruginosa
biofilms in cystic fibrosis. Future Microbiol 11:1663–1674
114. Barbara WT, Rabih OD (2004) Role of biofilm in catheter-
associated urinary tract infection. Am J Infect Control 3:177–183
115. James GA, Swogger E, Wolcott R, Ed P, Secor P, Sestrich J,
Costerton JW, Stewart PS (2008) Biofilms in chronic wounds.
Wound Repair Regen 16:37–44
116. Robert D. Kaplan. (2011) American interest: the wounded home
front American interest: http://www.cfr.org/world/american-
interest-wounded-home-front/p23550.Accessed 1 June 2015
11 7 . G h a f o o r A , H a y I D , R e h m H A B ( 2 0 11 ) R o l e o f
Exopolysaccharides in Pseudomonas aeruginosa Biofilm
Formation and Architecture. Appl Environ Microbiol 77:5238–
5246

118. Stoodley P, Sauer K, Davies DG, Costerton JW (2002) Biofilms as
complex differentiated communities. Ann Rev Microbiol 56:187–
209
119. McDougald D, Rice SA, Weichart D (2011) Non culturability:
adaptation or debilitation. Microb Ecol 25:1–9
120. Mah T, Toole GA (2001) Mechanisms of biofilm resistance to
antimicrobial agents. Trends Microbiol 9:34–39
121. Fazli M, Bjarnsholt T, Moller K (2011) Quantitative analysis of
the cellular inflammatory response against biofilm bacteria in
chronic wounds. Wound Repair Regen 19:387–391
122. Taj Y, Essa F, Aziz F, Kazmi SU (2012) Study on biofilm-forming
properties of clinical isolates of Staphylococcus aureus. J Inf Dev
Ctries 5:403–409
123. Attinger C, Wolcott R (2012) Clinically addressing biofilm in
chronic wounds. Adv Wound Care 1:127–132
124. Carlos J et al (2013) Biofilm formation by clinical isolates and the
implications in chronic infections. Biomed Cent Inf Dis 13:47
125. Schaber JA et al (2004) Analysis of quorum sensing-deficient
clinical isolates of Pseudomonas aeruginosa. J Med Microbiol
53:841–853
126. Harris LG, Murray S, Pascoe B, Bray J, Meric G, Mageiros L et al
(2016) Correction: biofilm morphotypes and population structure
among Staphylococcus epidermidis from commensal and clinical
samples. PLoS ONE 11(4)
127. Lawrence JR, Korber DR, Hoyle BD, Costerton JW, Caldwell DE
(1991) Optical sectioning of microbial biofilms. J Bacteriol 173:
6558–6567
128. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M,
Ersboll BK et al (2000) Quantification of biofilm structures by
the novel computer program COMSTAT. Microbiology 146:
2395–2407
129. Murga R, Stewart PS, Daly D (1995) Quantitative analysis of
biofilm thickness variability. Biotechnol Bioeng 45:503–510
130. Beyenal H, Donovan C, Lewandowski Z, Harkin G (2004) Three-
dimensional biofilm structure quantification. J Microbiol Methods
59:395–413
131. Wolcott RD, Rhoads DD (2008) A study of biofilm-based man-
agement in subjects with critical limb ischaemia. J Wound Care
17:145–155
K. Rahim et al.
132. Costerton JW, Stewart PS (2001) Antibiotic resistance of bacteria
in biofilms. Lancet 58:135–138
133. Donlan RM, Costerton W (2002) Biofilms: survival mechanisms
of clinically relevant microorganisms. Clin Microbiol Rev 15:
167–193
134. Stewart PS, Davison WM, Steenbergen JN (2009) Daptomycin
rapidly penetrates a Staphylococcus epidermidis biofilm.
Antimicrob Agents Chemother 53:3505–3507
135. Lewis K (2001) Riddle of biofilm resistance. Antimicrob Agents
Chemother 45:999–1007
136. Gilbert S, Loranger A, Daigle N, Marceau N (2002) Simple epi-
thelium keratins 8 and 18 provide resistance to Fas-mediated ap-
optosis. The protection occurs through a receptor-targeting modu-
lation. J Cell Biol 154:763–774
137. Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers
AK (2005) The exopolysaccharide alginate protects Pseudomonas
aeruginosa biofilm bacteria from IFN-γ-mediated macrophage
killing. J Immunol 175:7512–7518
138. Rani SA, Pitts B, Beyenal H, Veluchamy RA, Lewandowski Z,
Davison WM, Buckingham-Meyer K, Stewart PS (2007) Spatial
patterns of DNA replication, protein synthesis, and oxygen con-
centration within bacterial biofilms reveal diverse physiological
states. J Bacteriol 189:4223–4233
139. Hennequin C, Robin F, Cabrolier N, Bonnet R, Forestier C (2012)
Characterization of a DHA-1-producing Klebsiella pneumoniae
strain involved in an outbreak and role of the AmpR regulator in
virulence. Antimicrob Agents Chemother 56:288–294
140. Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004)
Specialized persister cells and the mechanism of multidrug toler-
ance in Escherichia coli. J Bacteriol 186:8172–8180
141. Roberts ME, Stewart PS (2005) Modeling protection from antimi-
crobial agents in biofilms through the formation of persister cells.
Microbiologyl 151:75–80
142. Akinobu I, Asami T, Thithiwat M, Kawata K, Okabe S (2009)
Increased antibiotic resistance of Escherichia coli in mature
biofilms. Appl Environ Microbiol 34:4093–4100
143. http://www.whasa.org/C_Region_ECapeNewsletter. Accessed 20
June 2016