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Recent Clinical Techniques, Results,
and Research in Wounds

Melvin A. Shiffman
Mervin Low Editors

Biofilm, Pilonidal
Cysts and Sinuses
Recent Clinical Techniques,
Results, and Research in Wounds

Series Editors

Melvin A. Shiffman
Mervin Low

More information about this series at http://www.springer.com/series/15695

Melvin A. Shiffman • Mervin Low
Editors

Biofilm, Pilonidal Cysts

and Sinuses
Editors
Melvin A. Shiffman Mervin Low
Tustin, CA Newport Beach, CA
USA USA

ISSN 2524-4590            ISSN 2524-4604 (electronic)

Recent Clinical Techniques, Results, and Research in Wounds
ISBN 978-3-030-03076-6    ISBN 978-3-030-03077-3 (eBook)
https://doi.org/10.1007/978-3-030-03077-3

Library of Congress Control Number: 2019932845

© Springer Nature Switzerland AG 2020

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,
and transmission or information storage and retrieval, electronic adaptation, computer software,
or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are
exempt from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in
this book are believed to be true and accurate at the date of publication. Neither the publisher nor
the authors or the editors give a warranty, express or implied, with respect to the material
contained herein or for any errors or omissions that may have been made. The publisher remains
neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Foreword1

It is a great honour for me to be invited to provide a foreword for the series of

six books edited by Dr. Shiffman and Dr. Low, which cover a broad expanse
of subjects relevant to and important in the care of patients with wounds.
Wounds have existed since the beginning of time and, until recent years,
have received scant attention unless major conflicts developed which neces-
sitated innovation in the treatment of patients with wounds. However, in
recent years there has been an increasing interest in this subject as evidenced
by the explosion of journals, meetings, societies and associations and initia-
tives that have been developed in this field.
The need for an academic underpinning of the subject of wound healing is
without question. Research papers published in recent years have undoubt-
edly enhanced the scientific basis for wound healing. This, coupled with
demographic changes in many countries around the world, has led to increas-
ing numbers of patients developing wounds or wound healing problems. It is
recognised that in the vast majority of geographies globally the number of
patients with wounds is increasing in everything other than major burns
where better health and safety initiatives have been an effective preventive
strategy.
This series of books not only attempts to deal with subjects that are nor-
mally seen in wound healing text but also provides a huge amount of space to
the management of wounds seen in surgical practice, both general and spe-
cialist surgery. The sections on infection are an attempt to deal with a very
common but poorly managed clinical problem and one that requires urgent
attention in view of the global challenge of antimicrobial stewardship. The
tradition chronic wounds are also included and provide a medical as well as a
nursing and paramedical focus on these subjects.
It is particularly pleasing to see books and chapters focused on specialised
surgical practice as these are areas that are rarely covered in other educational
products in this area. The opportunity for new therapies, measuring the range
of effective and appropriate outcomes and the use of new technologies are all
included.
For those of us who work in the area of wound healing, these books will
unquestionably be an important reference source. For those readers who want
to get an insight into this common, expensive and complex problem they will
without doubt find the content of these books an important source of informed
opinion and refer to the rapidly expanding evidence base that is developing in
this subject area.

v
vi Foreword1

I would urge you to immerse yourself in these books. Read, reflect and con-
sider how information that you have had access to can and will change your
clinical practice.

Keith Harding
Clinical Innovation Cardiff (ClIC),
College of Biomedical and Life Sciences,
Cardiff University School of Medicine,
Heath Park, Cardiff,
UK

1
P. S.
We, Melvin A. Shiffman and Mervin Low, are greatly enthralled by Keith Harding’s will-
ingness to write the Foreword for the books on wounds. Keith Harding is the Director of
TIME Institute (Translation, Innovation, Methodology and Engagement) and Head of the
Wound Healing Research Unit in the School of Medicine at Cardiff University. He is
Clinical Lead for Wound Healing in the Cardiff and Vale NHS Trust. In September 2013
Harding was appointed Dean of Clinical Innovation at Cardiff University. From 2002 to
2005 he was Head of the Department of Surgery at Cardiff University. He is Editor-in-
Chief of the International Wound Journal. Harding is a Past President of the European
Tissue Repair Society. He was the first President of the European Pressure Ulcer Advisory
Panel and first Recorder of the European Wound Management Association. He was Chair
of the International Working Group on Wound Healing in Diabetic Foot Disease in 2003.
He was Chair of the Expert Working Group that produced a range of International
Consensus Documents from 2004 to 2011. Professor Harding was appointed a Commander
of the Order of the British Empire in the 2013 New Year Honours for services to medicine
and healthcare.
Preface

We are delighted to have the book on wounds extended into six volumes.
There is so very much medical literature in journals and books that to cover
the whole gamut of wounds would be virtually impossible. We tried to include
as many of the experienced practitioners in wound care as possible, but many
of them are too busy to spend the time committing to submitting a chapter.
The selection of topics in each of the volumes was decided by the number
of authors responded to each of the subjects. As usual in editing a book, many
authors who agreed to submit manuscripts finally were not available to com-
plete the chapters. We contacted or tried to contact over 1500 authors and
most of them did not respond or the responses were not as good as expected.
The volumes include:

1. Biofilm, Pilonidal Cysts and Sinuses

2. Burns, Infections and Wound Management
3. Pressure Injury, Diabetes and Negative Pressure Wound Therapy
4. Plastic and Thoracic Surgery, Orthopedics and Ophthalmology
5. Vascular Surgery, Neurosurgery, Lower Extremity Ulcers, Antimicrobials,
Wound Assessment, Care, Measurement and Repair
6. Chronic Wounds, Wound Dressings and Wound Healing

There are many expert international contributors who have worked in vari-
ous aspects of wound research as well as clinical practice. We have tried to
have chapters that involved humans and in vivo results and avoided as much
as possible animals and in vitro results. Chapter conclusions are those of the
authors and may not be the same as those of the editors. At times the chapter
may appear cumbersome, but the authors try to show some proof of their
results. Language difficulties are common when translated into English so
that grammar, spelling and sometimes words have to be corrected.
Hopefully, the reader will get information that adds to their care and treatment
of patients. Researchers may gain knowledge of other researchers’ progress and
improve on the results or can continue their work in other directions. Controversy
is many times a good thing since looking in other directions to prove or disprove
a result can improve knowledge. We have a long way to go to be able to treat all
wounds properly and successfully in as short a time as possible.

Tustin, CA, USA Melvin A. Shiffman

Newport Beach, CA, USA Mervin Low

vii
Contents

Part I Biofilm

Biofilm: History, Cause, and Treatment. . . . . . . . . . . . . . . . . . . . . . 3

Melvin A. Shiffman
Biofilm: Clinical Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Tanja Planinšek Ručigaj
Mixed-Species Biofilm Compromises Wound
Healing by Disrupting Epidermal Barrier Function. . . . . . . . . . . . 21
Terri A. Zomerlei and Gayle M. Gordillo
Anti-biofilm Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Oana Săndulescu and Mihai Săndulescu
Wound Dressing in the Oral Cavity . . . . . . . . . . . . . . . . . . . . . . . . . 55
Esi Sharon, David Polak, Shay Sharon, and Nurit Beyth
Bacterial Biofilms on Wounds, a Major Factor
That Delays Wound Healing and a Potential Threat
to Human Life and Economy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Theerthankar Das, Onder Kimyon, and Michael J. Manefield
Antibiofilm Efficacy of Honeybee Products Against
Wound Biofilm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Martin Sojka, Miroslava Horniackova, Marcela Bucekova,
Viktor Majtan, and Juraj Majtan
Biofilm in Infective Endocarditis and Clinical Implications. . . . . . 109
Haytham Elgharably, Syed T. Hussain, Nabin K. Shrestha,
and Gosta B. Pettersson
Bacteriophage-Mediated Biocontrol of Wound Infections,
and Ecological Exploitation of Biofilms by Phages. . . . . . . . . . . . . 121
Stephen T. Abedon

Part II Pilonidal Cysts and Sinuses

History and General Information on Pilonidal

Cysts and Sinuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Melvin A. Shiffman

ix
x Contents

Pilonidal Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Sunil Anand and Sushila Chauhan
Pilonidal Sinus Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Dietrich Doll
Risk Factors of Pilonidal Sinüs in Teenagers. . . . . . . . . . . . . . . . . . 197
Turan Yildiz and Zekeriya Ilce
Wounds After Excision of Pilonidal Sinus Disease. . . . . . . . . . . . . . 203
Johannes Jongen and Volker Kahlke
Sclerosing Pilonidal Sinus Tracts by Crystallized
or Liquid Phenol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Cuneyt Kayaalp, Ersin Gundogan, and Metin Kement
Sacrococcygeal Pilonidal Sinus Disease . . . . . . . . . . . . . . . . . . . . . . 215
Aly Saber and Emad K. Bayumi
The Infected Pilonidal Sinus – Comparison of Conservative
versus Plastic Surgical Treatment after Excision. . . . . . . . . . . . . . . 231
Sonja Dahmann, Patricia Beatrice Lebo, and Max Vinzenz
Meyer-Marcotty
Minimally Invasive Surgical Approach to Complicated
Recurrent Pilonidal Sinus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Vahit Onur Gul, Sebahattin Destek, and Serkan Ahıoglu
Treatment of Pilonidal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Barış Sevinç
Tandem Rhomboid Flap Repair: A New Technique in
Treatment of Extensive Pilonidal Disease of the Natal Cleft. . . . . . 255
Naveen Narayan and Prema Dhanraj
Conservative vs. Surgical Interventions for
Umbilical Pilonidal Sinus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Mehmet Kaplan, Huseyin Cahit Yalcin, Onder Ozcan,
and Fatma Cigdem Kaplan
Subcutaneous Destruction of Sinus Tract with
Long-Term Vessel-Loop Drainage as Minimal
Invasive Surgical Treatment for Primary Pilonidal Sinus. . . . . . . . 277
Michael Korenkov
Correction to: Treatment of Pilonidal Disease. . . . . . . . . . . . . . . . . 283
 Part I
Biofilm
 Biofilm: History, Cause,
and Treatment

Melvin A. Shiffman

1 Introduction removed from vinegar barrels known as “mère du

vinaigre” [4].
Biofilm is a very persistent problem in chronic Originally described as “slime” or “film,” the
wounds. The bacteria grouping is protected from terms were referring to bacterial adhesion, aggre-
the effect of antibiotics by a polymer film cover- gation, and multiplication on surfaces. The earli-
ing. Antibotics cannot penetrate that film and est use of “biofilm” was by ZoBell and Allen [5]
destroy the offending organisms. Debridement who studied the adherence and growth of bacteria
followed by antibiotics has been the only method on submerged glass slides in sea water. They
to treat chronic wounds. Recently antibiofilm found that fouling was initiated by biofilm-­
products have been identified that will uncover growing bacteria and, to a lesser extent, other
the organisms and allow antibiotics to work. microorganisms and that such films favor the
subsequent attachment of the larger and more
inimically fouling organisms.
2 History A publication by Mack et al. [6] had an
abstract that read: “After the deposit of a small
On April 24, 1676, van Leeuwenhoek examined amount of debris upon a hard surface, the bacte-
his steeped-pepper preparation with a micro- rial cells attach and develop the matrix on which
scope and with “great wonder” (groote verwon- the biofilm is formed. The transmission and scan-
dering) observed several kinds of microorganisms ning electron microscopes were used to visualize
in the water, including what are now called bacte- the sequence of the biofilm development in the
ria [1]. The report of the discovery of bacteria trickling wastewater filter.”
was contained in a letter sent to the Royal Society Jendresen and Glantz [7] used the term bio-
and dated at Delft on 9 October 1676 [2]. film in dental circumstances. Serralta et al. [8]
Pasteur observed and sketched aggregates of hypothesized that biofilms do exist in wounds.
bacteria as the cause of wine becoming acetic, “Biofilms, like other communities, form gradu-
which led to his discover of pasteurization [3]. ally over time. In order for a biofilm to form, bac-
Pasteur described Mycoderma aceti as the caus- teria must be able to attach to a substrate. This
ative agent in “la matière visqueuse,” a membrane attachment is largely based on nutritional signals
and a critical number of organisms assembling.
Once attached, the bacteria relinquish their
planktonic state and begin to recruit other bacte-
M.A. Shiffman, M.D., J.D.
17501, Chatham Drive, Tustin, CA 92780-2302, USA ria.” The aggregates proliferate and recruit new
e-mail: [email protected] members that can be of different species of

Recent Clinical Techniques, Results, and Research in Wounds (2017) 3

DOI 10.1007/15695_2017_1, © Springer International Publishing AG
Published Online: 24 October 2017
4 M.A. Shiffman

b­ acteria, fungi, or protozoa. “Attached bacteria Molecular diagnostics are available to provide
excrete an extracellular polysaccharide matrix, comprehensive, rapid, and accurate microbial
which forms the structural architecture of the detection and quantification of previously
biofilm.” The biofilm colonies are constantly unidentifiable organisms, including yeast and
changing and adapting to their environment. fungi. However, although major resistance fac-
tors are also elucidated, classical species suscep-
tibility is not provided. Wolcott et al. [16]
3 Cause reported that they were able to increase complete
closure rates from 48 to 62% over a 6-month
Given the right conditions, all bacteria can grow a study period utilizing multiple concurrent strate-
biofilm. Biofilm forms when microbial cells gies including frequent debridement and molecu-
attach to a hard surface or lining tissue and evolve lar diagnostics to guide systemic antibiotic
into a microbial community encased within a self- intervention. As a result, systemic antibiotic
produced polymer matrix. Biofilm bacteria are usage increased from 32 to 67% of patients
less susceptible to the immune system and can receiving therapy. Although there was an overall
persist for long periods of time. Phagocytes have increase in antibiotic usage, the increased utiliza-
difficulty ingesting bacteria within a biofilm due tion remains in alignment with the literature for
to the antiphagocytic properties of the biofilm this patient population [17, 18].
matrix [8, 9]. Biofilms display innate resistance to Dowd et al. [19] reported significantly
antimicrobial agents [10]. Biofilms increase the increased rates of wound closure with the multi-
opportunity for gene transfer between and among ple concurrent strategies of biofilm-based wound
bacteria and can convert previous avirulent organ- care and the combination of individualized topi-
isms into highly virulent pathogens [11]. cal antibiofilm therapy guided by molecular
Antibiotics do not penetrate the biofilm matrix diagnostics.
[11, 12]. It is possible that a biofilm-specific phe- Many antibiofilm compounds have been iden-
notype may be induced in a subpopulation of the tified by Rabin et al. [20] from diverse sources.
biofilm [11] and these subpopulations express
active mechanisms to reduce the efficacy of anti- 1. Natural sources
biotics [13, 14]. (a) Brominated furanones
(b) Garlic
(c) Ursine triterpenes
4 Treatment (d) Corosolic acid and asiatic acid
(e) Ginseng and 3-indolylacetonitrile
Treatment for biofilm is to disrupt and removing
the biofilm by debridement of the wound. After 2. Imidazole derivatives
this, specific antibiotic treatment can be (a) Bromoageleferin and oroidin, which were
instituted. isolated from the sponge
Molecular diagnostics provide the first objec-   Agelas conifer
tive means of assessing wound bioburden. The
accuracy and comprehensive data from such 3. Indole derivatives
diagnostic methodologies provide clinicians with (a) Resveratrol 3-Indolylacetonitrile
the ability to employ patient-specific treatment (b) Indole–triazole-amide analogs
options, targeted to each patient’s microbial (c) Benzimidazoles analogs
wound census [15]. Based on current outcomes
data, the most effective therapeutic options are 4. Plant-derived compounds
topical antibiofilm agents combined with topical (a) Emodin
antibiotics. In specific patients, systemic antibiot- (b) Phloretin
ics and selective biocides are also appropriate. (c) 7-Epiclusianone
Biofilm: History, Cause, and Treatment 5

(d) Isolimonic acid 4. Pasteur L (1864) Memoire de la fermentation acé-

tique. Ann Scient de L'ÉNS I serie, Tome I Gauthier
(e) Casbane diterpene
Villars, pp. 113–58
(f) Chelerythrine 5. ZoBell CE, Allen E (1935) The significance of marine
(g) Hyperforin and its hydrogenated analog bacteria in the fouling of submerged surfaces.
(h) Proanthocyanidin A2-phosphatidylcholine J Bacteriol 29:239–251
6. Mack WN, Mack JP, Ackerson AO (1975) Microbial
(i) Ellagic acid
film development in a trickling filter. Microb Ecol
(j) Ellagic acid mannopyranoside 2(3):215–226
(k) Ellagic acid xylopyranoside 7. Jendresen MD, Glantz PO (1981) Clinical adhesive-
(l) Ginkgoneolic acid ness of selected dental material. An in-vivo study.
Acta Odontol Scand 39(1):39–45
(m) Tannic acid
8. Serralta VW, Harrison-Balestra C, Cazzaniga AL,
(n) (R)-Norbgugaine Davis SC, Mertz M (2001) Lifestyles of bacteria in
(o) Ginkgolic acid C15:1 wounds: presence of biofilms? Wounds 13(1):29–34
9. Johnson GM, Lee DA, Regelmann WE, Gray ED,
Peters G, Quie PG (1986) Interference with granulo-
5. Marine-derived compounds
cyte function by Staphylococcus epidermidis slime.
(a) Auromomycin Infect Immun 54(1):13–20
(b) Halogenated furanones 10. Evans RC, Holmes CJ (1987) Effect of vancomycin
(c) Brominated alkylidene lactams hydrochloride on Staphylococcus epidermidis bio-
film associated with silicone elastomer. Antimicrob
(d) Bromopyrrole alkaloids
Agents Chemother 31(6):889–894
11. Lewis K (2001) Riddle of biofilm resistance.
6. AHLs-based inhibitors Antimicrob Agents Chemother 45(4):999–1007
(a) N-Acyl homoserine lactones 12. Shigeta M, Tanaka G, Komatsuzawa H, Sugai M,
Suginaka H, Usui T (1997) Permeation of antimicrobial
(b) Cationic peptides
agents through Pseudomonas Aeruginosa biofilms: a
(c) Cathelicidin peptide LL-37 simple method. Chemotherapy (Tokyo) 43(2):340–345
(d) d-Amino acids 13. Gilbert P, Das J, Foley I (1997) Biofilms susceptibility
to antimicrobials. Adv Dent Res 11(1):160–167
14. Maira-Litrán T, Allison DG, Gilbert P (2000) An
Conclusions evaluation of the potential of the multiple antibiotic
resistance operon (mar) and the multidrug efflux
There are now some measures besides debride-
pump acrAB to moderate resistance towards cipro-
ment to attack the biofilm in chronic wounds floxacin in Escherichia coli biofilms. J Antimicrob
allowing access for antibiotics to reach the Chemother 45(6):789–795
infective bacteria in the wound. It behooves 15. Jones CE, Kennedy JP (2012) Treatment options
to manage wound biofilm. Adv Wound Care (New
those physicians treating wounds to become
Rochelle) 1(3):120–126
familiar with antibiofilm agents. 16. Wolcott RD, Cox SB, Dowd SE (2010) Healing and
healing rates of chronic wounds in the age of molecu-
lar pathogen diagnostics. J Wound Care 19(7):272–
278. 280–1
17. Tammelin A, Lindholm C, Hambraeus A (1998)
References Chronic ulcers and antibiotic treatment. J Wound Care
7(9):435–437
1. Bardel D (1982) The roles of the sense of taste and 18. Howell-Jones RS, Price PE, Howard AJ, Thomas DW
clean teeth in the discovery of bacteria by Antoni van (2006) Antibiotic prescribing for chronic skin wounds
Leeuwenhoek. Microbiol Rev 47(1):121–126 in primary care. Wound Repair Regen 14(4):387–393
2. van leeuwenhoek A. Letter of 9 October 1676 to the 19. Dowd SE, Wolcott RD, Kennedy J, Jones C, Cox SB
Royal Society, London. Royal Society, MS. L 1. 22 (2011) Molecular diagnostics and personalised medi-
3. Pasteur L (1922) Memoire sur la fermentation ace- cine in wound care: assessment of outcomes. J Wound
tique. Ann Scient L'Ecole Normale Superiure. In: Care 20(5):232, 234–9
Oeuvres des Pasteur, réunies par Pasteur Valerie-­ 20. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu
Radot, Tome II. Fermentations et generations dites E, Sintim HO (2015) Agents that inhibit bacterial bio-
spontanees. Masson, Paris film formation. Future Med Chem 7(5):647–671
 Biofilm: Clinical Experience

Tanja Planinšek Ručigaj

1 Introduction exposed in an aqueous medium will almost imme-

diately become coated by polymers from that
Biofilm is an assemblage of microbial cells which medium. That will result in chemical modification
have an extracellular polysaccharide matrix on a and affect the rate and extent of microbial attach-
surface [1, 2]. In the 1650s van Leeuwenhoek dis- ment [9]. Loeb and Neihof were found that bio-
covered the biofilm. In 1978 research on biofilm films were formed within minutes and grow for
has exploded [3]. In the last decade the concept of several hours [10]. Tolker-Nielsen and Molin [11]
biofilm in clinical practice was emerged [3]. noted that every microbial biofilm community is
The development of infection delays the nor- unique. Bacteria from the oral cavity colonize pel-
mal wound healing process [4, 5]. Endotoxin and licle-conditioned surfaces within hours of exposure
exotoxin produced by the microorganisms and [12]. Blood, saliva, tears, intervascular fluid, urine,
local inflammatory processes cause the wound and respiratory secretions influence the attachment
infection signs: pain, redness, swelling, pain and of bacteria to biomaterials [13].
odor or pus [6–8], and fragile, brown granulation. Nutrient levels, ionic strength, pH, and tempera-
Biofilms play a significant role of infections spe- ture of aqueous medium have an important role of
cial at chronic wounds too [1]. microbial attachment to a substratum. An increase in
Most often bacteria are found in two forms: like the concentrations of ferric iron, sodium, and cal-
sessile biofilm cells and like free-flowing bacteria cium is important to attachment of Pseudomonas
in suspension—planktonic form which is rare [3]. fluorescens to glass surfaces (they represent repul-
The solid-liquid interface is an ideal environment sive forces between the glass surface and the nega-
for the attachment and growth of microorganisms tively charged bacterial cells) [14]. The hydrophobic
(water, blood). The solid surface which is cell surface of microbial cells, fimbriae, flagella, and
hydrophobic, nonpolar (Teflon, plastics) attaches extracellular polymeric substance matrix influence
more rapidly the microorganisms than hydrophilic the rate of attachment [9]. Amino acids from fim-
materials (glass, metals). A material surface briae contribute to hydrophobicity of cell surface
and that influence initial electrostatic repulsion bar-
rier too [15].
The biofilm on water system contains filamen-
T.P. Ručigaj, M.D. tous bacteria, freshwater diatoms, and corrosion
University Medical Centre Ljubljana,
Ljubljana, Slovenia products with clay. On the other hand the bio-
films on the medical device are composed from
Dermatovenerological Clinic, University Clinical
Centre Ljubljana, Ljubljana, Slovenia one species of bacteria with extracellular poly-
e-mail: [email protected] meric substance matrix [9].

Recent Clinical Techniques, Results, and Research in Wounds (2017) 7

DOI 10.1007/15695_2017_2, © Springer International Publishing AG
Published Online: 24 October 2017
8 T.P. Ručigaj

2 Forming of Biofilm against antibiotics, disinfectants, and certain pro-

cedures of debridements, but also protects the
Stages of development of biofilms are as follows: bacteria from other external influences. Coat is
permeable to oxygen from the environment, and
1. Reversible fastening of the freely floating bac- it allows the passage of carbon dioxide into the
teria on the surface. environment. Coat is permeable to nutrients. The
2. Irreversible fastening, which allows the creation bacteria dismiss the degradation products in the
of communities or colonies of bacteria. Bacteria neighborhood through a special water channel.
with fimbriae or flagels surface can easier be Bacteria in biofilm metabolize more slowly,
attached [15]. They can attach to different surfaces reproduce less frequently, and show different
(like medical devices, plumbing system) phenotype traits than the same planktonic bacte-
(corrosion) and to living tissue (wounds, teeth). ria. Bacteria in biofilm are 1000 times more resis-
Biofilm is formed on places where liquid and tant to antimicrobial therapy (as opposed to
solid media surface comes together [9]. The planktonic bacteria) [21].
easiest it sticks to is on hydrophobic, nonpolar 4. So a mature biofilm is formed. Quorum-­
surface [16]. Gram-­positive bacteria that secrete sensing molecules (pheromones) manage with
myolitic acid are easier to attach to the the planktonic and sessile microorganism [22].
hydrophobic substrate, while the extracellular 5. In the next stage, there is a partial decomposi-
polymeric substances and lipopolysaccharide (O tion of the biofilm with the help of various
antigens) of Gram-­negative bacteria are important enzymes. The greater is the flow of liquid on
in attaching onto the hydrophilic substrate the surface of the biofilm and the modification
(Table 1) [9, 17, 18]. Even the regulation of genes of the base on which they are fastened bacteria,
encoding enzymes involved in glycolysis or the faster is the decomposition of a biofilm,
fermentation (phosphoglycerate mutase, alcohol and the release of individual bacteria [23].
dehydrogenase, and triosephosphate isomerase) 6. Individual bacterial cells are re-released into
of Staphylococcus aureus influences biofilm the environment.
formation [19]. Genes that control the synthesis 7. Individual free-floating bacteria find new
of polyphosphokinase and algD, algU, and rpoS areas for settlement.
are important in the formation of biofilm of 8. New re-formed colonies are protected by poli-
Pseudomonas aeruginosa [20]. meric matrix (slime) and biofilm occurs
3. Bacteria grow quickly in the colonies under a (Fig. 1).
protective matrix, which makes it even easier to 9. We measure speed of creating of biofilm in hours.
reproduce and provides a higher survival rate. The acceptance of microbes to the surface lasts
The matrix is largely composed of polysaccha- only a minute. The growth of microcolonies takes
rides from Gram-negative bacteria (mostly neu- a 2–4 h. The development of the initial extracellular
tral or polyanionic). The presence of d-glucuronic, polysaccharide matrix lasts 6–12 h. The mature
d-galacturonic, and mannuronic acid or ketal tied biofilm is constructed in 2–4 days, depending on
pyruvates decide on anionic properties [15]. the species and growth conditions [24].
These properties enable the calcium and magne-
sium divalent cation binding easier. Polymer Some of the frequent microorganisms which
extracellular matrix not only protects the bacteria make a biofilm on the wounds are S. epidermidis,

Table 1 Important factors which influence cell attachment and biofilm formation [9]
Properties of the substratum Properties of the bulk fluid Properties of the cell
Texture or roughness Flow velocity Cell surface hydrophobicity
Hydrophobicity pH Fimbriae
Conditioning film Temperature Flagella
Cations Extracellular polymeric substances
Presence of antimicrobial agents
Biofilm: Clinical Experience 9

S. aureus, and P. aeruginosa [25, 26]. Biofilms 3 Identification of Biofilm

can be found in 6% on acute wounds and in 60%
on chronic wounds [23, 27]. Indirect clinical indicator of biofilm in the wound
Pseudomonas aeruginosa grow primarily as a is shiny, slough, devitalized fibrin which is
base of biofilm and growth faster as a pure culture opaque loosely attached in same parts of the
in biofilm than in the mixed culture. It rapidly col- wound bed (Table 2) (Fig. 2) [23, 30]. It was a big
onized the surface K. pneumoniae form localized problem to prove the biofilm formation in wounds
microcolonies (covering around 10% of the area) because biofilms are very small size and wound
so they have greater access to oxygen and nutrients biofilm bacteria are difficult or impossible to cul-
[28]. Gilbert et al. [29] showed that hydrophobic- ture [31]. Now identification of biofilm formation
ity of newly dispersed cells is low and increases is possible by light or electron microscopy and
with continued incubation and growth. confocal laser ­scanning microscopy and using

Unattached cells

Matrix synthesis

Attached cells

Adherence Colonisation Biofilm formation

Fig. 1 Forming of biofilm

Table 2 Indirect clinical indicator of biofilm

Clinical observation Presence of biofilm
The material from surface can gently and without trauma blown away from the surface by +
applying techniques of physical removal (swab, pads, sharp debridement)
Excessive moisture +
Friable hypergranulations +
Quickly (within a day or two) reformed surface material without frequent interventions +
(cleansing, debridement)
Signs of local infection: redness, heat, swelling, pain, odor +
Recurring infection is present despite antimicrobial therapy: antiseptics (silver, PHMB, +
DACC, iodine), antibiotics
The linger surface material is present at wound bed despite autocatalytic or enzymatic +
debridement
Recalcitrant wound despite treating underlying co-morbidities +
Reacts biofilm on the multimodal strategy as debridement, cleaning, and antimicrobial +
dressings with silver
10 T.P. Ručigaj

Visual indicators

Suface substance detach

easily and atraumatically

Yes
No

Probably Suface substance persist

BIOFILM despite of autolytic or enzymatic
with debridment
increasing Probably host
confidence Yes No devitalised
tissue
Suface substance re-form quickly
without of frequent intervention

Yes No

Indirect indicators

Wound poorly respond

to antibiotics

Yes No
Probably
Wound poorly respond planktonic
to antseptics bacteria
Yes
No

Wound respond to multi-modal therapy Underlying

comorbidity
Yes No

Fig. 2 The flowchart of clinical algorithm of biofilm in the wound

the fluorescent dye [23, 32–37]. Extracellular Different studies have shown that treatment
polymeric, polysaccharide matrix which sur- of adsorbed cells with proteolytic enzymes
rounds the bacteria in biofilm can be demon- caused a release of attached bacteria [40, 41].
strated with staining with ruthenium red, The antibiotic tolerance of the biofilm in vitro
carbohydrate stains, and concanavalin A [38]. studies shows that the biofilm can withstand
treatment with very high dosages of antibiot-
ics. It can be up to 1000 times higher than the
4 Biofilm Response minimal inhibitory concentration is [42]. The
to Different Treatments biofilm matrix from proteins, extracellular
DNA, and polysaccharides reduces penetra-
For chronic wounds to begin to heal, we need to tion and bind of the antibiotics [43–49].
remove the biofilm. The greatest role alongside For removing the biofilms, antiseptics are pre-
with sharp debridement is of biological and ultra- ferred over antibiotics [24, 50]. Silver and PHMB
sound debridement and changes in electrical are very effective against planktonic bacteria and
charge. Last but not least important are antisep- immature biofilms (Table 3). Applied on mature
tics, which can slightly slow down the formation biofilms they can only inhibit further growth and
of biofilm. Significant antimicrobials against bio- prevent bacteria from spreading beyond the bio-
film are silver and polyhexene-biguanides film but not resolve the infection [2, 51–53]. The
(PHMB) [3, 39]. antimicrobial efficacy of silver dressings against
Biofilm: Clinical Experience 11

Table 3 Antiseptics against biofilm

AMA GMS WUWHS EWMA
Antiseptic React on bacteria Acts on biofilm 2004 2007 2008 2013
Octenidine Pseudomonas aeruginosa EPS destruction, alteration + + + +
dihydrochloride Staphylococcus aureus of phospholipid, changes
Staphylococcus epidermidis in cell wall
Streptococcus spp.
Polyheksanide Pseudomonas aeruginosa Penetration through the + + + +
Staphylococcus epidermidis matrix biofilm, killing
Escherichia coli sessile bacteria
Povidone iodine Pseudomonas aeruginosa Effect on matrix + + + +
Staphylococcus aureus glycocalyx
Staphylococcus epidermidis
Nanocrystal silver Pseudomonas aeruginosa Destruction of the biofilm +
Staphylococcus aureus matrix, destruction of
Enterobacteriaceae sessile bacterium
Manuka honey Staphylococcus aureus Penetration through the + +
Pseudomonas aeruginosa matrix of the biofilm, kills
MRSA sessile bacteria

bacterial biofilms was investigated by in vitro and 5 Clinical Experience

in vivo models. Hegger and his colleagues show
that dressings with silver reduced biofilm less than In all studies we measured only the wound size
90% in 1-week treatment in an animal model [54]. and isolated bacteria, but did not search for bio-
But in vitro methodes show better results as proven film, because at that time we did not have the
by Percivaletal. They reported that Hydro fiber possibilities for that. All the studies included
dressings with silver acting against bacterial bio patients with the wounds that were lasting for a
films after 24 h of treatment, and total killed bacte- long time and the wound bed was slough, so
rial biofilm within 48 h [55]. there were big possibilities for biofilms on them.
Ultrasound waves can disrupt biofilm but the
decrease in bacterial counts was not significant [56–
63]. Bio-debridement with larvae and debridement 5.1  tudy 1: Honey and Metal
S
with negative pressure therapy also have positive Ions’ Positive Effects [70]
secondary effects on combining disruption of bio-
films [64–66]. But gold standard and most important 1. 60 venous leg ulcers (ABPI > 0.8) were ran-
and effective to remove biofilm is sharp debridement domly positioned into one of the two groups
which we repeated during 48–72 h. With that we by closed numbered envelopes.
allow the opening of the “therapeutic window” and 2. Patients were observed for 6 weeks or less (if
can apply the antiseptics [23]. Reformation of bio- ulcers were healed).
film after mechanical destruction is possible within 3. Exclusion criterias: Insulin-dependent diabe-
24 h, depending on species, from planktonic bacteria tes mellitus, rheumatoid arthritis, uncontrol-
from biofilm or growth from bacteria newly intro- lable hypertension, cardiac decompensation,
duced into the wound [24, 53, 67, 68]. carcinoma, and immobility.
Therapy against biofilm will be successful if we: 4. Including criterias: Ankle brachial pressure
index (ABPI) higher than 0.8.
1. Break down quorum-sensing molecules
5. Wound bed: Staging in B or C class by
2. Degrade extracellular polymeric substances
Falanga’s classification, and delayed or
3. Block acceptance of biofilm on surface
stopped healing, risk of infection, foul odor,
(Fig. 3) [23, 24, 69]
or discoloration of granulation tissue.
12 T.P. Ručigaj

Chronic wound
(no healing, antibiotics not work)

Reduction of biofilm

Fig. 4 Patients treated with honey before study

Energic debridement

Prevention of wound
recontamination
Antimicrobial dressings
Aseptic condition
Fig. 5 Patients treated with honey after study

9. Mean duration of ulcer in group 1 before

study was 27 months, while in group 2 that
Supression of formation of biofilm time was 38 months.
with apply topical antiseptics 10. At the beginning of the study, ulcers’ areas
were drawn into appointed film dressings
and precisely measured with a digital pla-
nimeter (Placom KP-90N; Japan); the latter
was repeated at the end as well.
Healing 11. As for the size of the ulcers in MelMax®
group they show a great reduction from
mean 28 cm2 (min. 1 cm2, max. 133 cm2) to
Fig. 3 The principles of operating on biofilm 17 cm2 (min. 0 cm2, max. 72 cm2), which
represents impressive 36.7% in mean dura-
tion of treatment of 44 days (Figs. 4 and 5).
6. First group, with mean age 72, was treated 12. In Actisorb® group they show stagnation with
with honey-based dressings (MelMax®). little or no improvement over time (mean
7. Second group was treated with silver/ duration of therapy was 42 days) from the
charcoal-­based dressings (Actisorb beginning mean size of 16 cm2 (min. 1 cm2,
silver®). max. 74 cm2) to the final 15 cm2 (min. 1 cm2,
8. Furthermore compression with long-stretch max. 70 cm2), which represents merely 2.8%
bandages was used at every patient. decrease in area (Tables 4 and 5).
 Biofilm: Clinical Experience 13

Table 4 Differences of wound sizes from both dressings

Wound size at the

36 start (cm2)
Wound size at the
125 end (cm2)

100

75 27 36
* 27 33

23
32
50 23

25

As - Actisorb MM - Melmax
Wound dressing

Table 5 Wound size reductions in both dressings in percentage

Wound size reduction percentage in research perios (%)

100

75

50

25

-25

AS - Actlsorb MM - Melmax
Wound dressing
14 T.P. Ručigaj

13. Because of the possible risk of systemic

infection each day one swab from the wound
bed was taken in 15 patients, 9 from group 1
and 6 from group 2. Of those in group 1 after
1 week of therapy with honey-based dressing
only one patient needed additional systemic
antibiotic therapy, while of those in group 2
after 1 week of therapy with silver/charcoal-­
based dressing all needed additional sys-
temic antibiotic therapy.

Fig. 6 Patients treated with alginate dressing with silver

5.2  tudy 2: The Effects of Alginate
S before study
Dressings with Silver
on Healing Rate and Pain

In a randomized study we evaluated the effects of

two alginate dressings on the healing:

1. Patients with 20 pain venous leg ulcers in

stage C3 were included.
2. They were randomly included into one of the
groups by closed numbered envelopes.
3. Compression with long-stretch bandage was
used in every patient. Fig. 7 Patients treated with alginate dressing with silver
4. Half of the ulcers were treated with alginate after study
dressings with silver Silvercel®. Table 7 Pain before and after study with different algi-
5. Other half of the ulcers were treated with nate dressings
another calcium alginate dressing Algisite M®. Before After Average days of
6. At the beginning and at the end of the study study therapy therapy/patient
the ulcer areas were drawn onto film dressing Silvercel® Severe No pains 16.0 days
and then precisely measured by using digital (10 ulcers)
planimeter (Table 6) (Figs. 6 and 7). Algisite M® Severe Moderate 15.5 days
(10 ulcers)
7. At the beginning and at the end of the study ques-
tionnaires, concerning pain was filled (Table 7).
was smaller at the patients treated with algi-
8. Only one of the four categories was possible to
nate dressings with silver (Table 7) [71].
choose (no, mild, moderate, and severe pain).
9. Results: Ulcers treated with alginate with sil-
ver (Silvercel®) were smaller for 15.4% and 5.3  tudy 3: Comparative
S
ulcers treated with calcium-alginate without Clinical Trial
silver were larger for 0.7% (Table 6) and pain
Comparing the hydrofiber dressing and ointments
Table 6 Wound size before and after study with different in changing microbial colonization and healing:
alginate dressings
Before After Average days 1. 24 patients with 42 venous leg ulcers (ABPI
study therapy of therapy/
(cm2) (cm2) patient
0.80–1.40; B 2–3)
Silvercel (10 ulcers) 130.6
®
110.5 16.0 days 2. Treated: 12 hydrofiber and 12 with ointments
Algisite M® (10 ulcers) 107.3 108.0 15.5 days 7 weeks
Biofilm: Clinical Experience 15

Fig. 10 Patients treated with dressing with PHMB before

study
Fig. 8 Patients treated with hydrofiber dressing before
study

Fig. 11 Patients treated with dressing with PHMB after

study

Fig. 9 Patients treated with hydrofiber dressing after Table 8 Wound bed and size before and after applying
study the dressing with PHMB
Before study At the end of the study
Wound bed C3 all A2 all
Results: The most frequently isolated bac-
cm2 24.44 17.69
teria in group with using hydrofibre-dressings
(Aquacel®) was P. aeruginosa (at the begin-
ning: 44.4%; at the end: 20%) in group with (mean). Foam AMD with PHMB® changed
ointment was most frequently P. aeruginosa every 3–4 days. The treatment lasted (mean)
(at the beginning: 53.3%; at the end: 60%). 44.7 days or until wound did not healed. Results:
Mean wound size at treating wounds with One wound healed. The others were smaller and
hydrofiber dressing was 9.6 cm2 at the begin- measurement at the end 17.69 cm2 mean (Figs. 10
ning and 8.8 cm2 at the end. Mean wound size and 11). The wound beds were in stage A2 in all
at the treating wounds with ointments was patients (Table 8) [73].
16.4 cm2 at the beginning and 19.5 cm2 at the
end (Figs. 8 and 9) [72].
5.5  tudy 5: Experience
S
with the First Slovenian
5.4  tudy 4: Wound Management
S Dressing for the Treatment
with Foam with PHMB of Chronic Infected Wounds

This small study included five patients with no In a randomized clinical study, effects were evalu-
progress in healing venous leg ulcers for ated on healing and pain in 14 venous leg ulcers
5.2 years (mean). At the beginning their wound with wound beds C2–3 treated with honey
beds were in stage C3 and size was 24.44 cm2 (Vivamel®—group 1) and 16 venous leg ulcers with
16 T.P. Ručigaj

wound bed C3 treated with antiseptic (AMD with

PHMB®—group 2). Results: In group 1 healing
was fasters (after one week), but patients in group 2
experienced less pain than patients in group 1. After
2 weeks all the ulcers from both groups were in B2
stage by Falanga’s classification of wound bed
(Table 9) (Figs. 12, 13, 14, and 15) [74].

Table 9 Wound bed and pain before and after applying

dressings with PHMB and honey Fig. 14 Patients treated with dressing with honey before
study
Pains
After After under the
Day 0 1 week 14 days dressings
Group 1 : C3 (5 B2 (11 B2 (11 At 4
Vivamel®, ulcers) ulcers) ulcers) patients
Tosama C2 (9 A2 (8 A2 (8
ulcers) ulcers) ulcers)
B2 (2
ulcers)
B3 (3
ulcers)
Group 2 : C3 (16 C2 (8 B2 (8 No body
AMD Foam, ulcers) ulcers) ulcers)
Covidien® B2 (8 A2 (8
ulcers) ulcers) Fig. 15 Patients treated with dressing with honey after study

5.6 Study 6: Treatment

with Different Alginate
Dressings

In a small clinical trial seven patients were

included (five women, two men; average age of
77.14 years) with venous leg ulcers (ABPI 0.8
and higher) in stage C3 (Falanga V. classification
of wound bed). Average time of treatment was
Fig. 12 Patients treated with dressing with PHMB before 4.43 days. Alginate dressings are used for
study sloughy wounds with fibrinous bed. One half of
ulcer was treated with calcium alginate dressing
with added manganese and zinc ions and chloro-
phyllin alginate dressing (Trionic®) and the other
half of the same ulcer with another alginate dress-
ing (Algisite M®). Compression with long-stretch
bandages was used at every patient. Results: Half
of the ulcer treated with calcium alginate dress-
ing with added manganese and zinc ions and
chlorophyllin alginate dressing showed better
progress in 100% whether the other half of the
same ulcer treated with another Ca alginate dress-
Fig. 13 Patients treated with dressing with PHMB after ing showed progress in only 28% (Table 10)
study (Figs. 16 and 17) [75, 76].
Biofilm: Clinical Experience 17

Table 10 Wound bed at the beginning and at the end of organized in biofilm formation significantly
the study affect the healing of chronic wounds.
In the In the end: In the end: Identification of biofilm is still no part of the
Patients beginning Trionic® Algisite M® daily routine. But the sharp debridment is still
1. C3 A2 C3 the most efficient option for removing the
2. C3 B3 C3
biofilm.
3. C3 B3 C3
4. C3 A2 B3
5. C3 A3 B3
6. C3 B3 C3
References
7. C3 B2 C3
1. Percival SL, Bowler PG (2004) Biofilms and
their potential role in wound healing. Wounds
16(7):234–240
2. Ručigaj TP (2016) Biofilm and our clinical experi-
ence. Acta Med Croatica 70:57–59
3. Alhede M, Alhede M (2014) The biofilm challenge.
Eur Wound Manage Assoc J 14(1):54–58
4. Loewenthal J (1962) Sources and sequelae of surgical
sepsis. BMJ 1:1437–1440
5. Cutting KF, Harding KG (1994) Criteria for identify-
ing wound infection. J Wound Care 3:198–201
6. Ovington L (2003) Bacterial toxins and wound heal-
ing. Ostomy Wound Manage 49(7A Suppl):8–12
7. European Wound Management Association (EWMA)
(2005) Position document: identifying criteria for
wound infection. MEP Ltd., London
Fig. 16 Patient during the study with different alginate 8. Metcalf DG, Parsons D, Bowler PB (2016)
dressings Development of a next-generation antimicrobial
wound dressing. Acta Med Croatica 70:49–56
9. Donlan RM (2002) Biofilms: microbial life on sur-
faces. Emerg Infect Dis 8(9):881–890
10. Loeb GI, Neihof RA (1975) Marine conditioning
films. Adv Chem 145:319–335
11. Tolker-Nielsen T, Molin S (2000) Spatial organiza-
tion of microbial biofilm communities. Microb Ecol
40:75–84
12. Marsh PD (1995) Dental plaque. In: Lappin-­
Scott HM, Costerton JW (eds) Microbial biofilms.
Cambridge University Press, Cambridge, pp 282–300
13. Mittelman MW (1996) Adhesion to biomaterials. In:
Fletcher M (ed) Bacterial adhesion: molecular and
ecological diversity. Wiley-Liss, Inc., New York,
pp 89–127
14. Fletcher M (1988) Attachment of Pseudomonas
fluorescens to glass and influence of electrolytes on
bacterium-substratum separation distance. J Bacteriol
170:2027–2030
15. Corpe WA (1980) Microbial surface components
Fig. 17 Patient at the end of the study with different algi- involved in adsorption of microorganisms onto sur-
nate dressings faces. In: Bitton G, Marshall KC (eds) Adsorption
of microorganisms to surfaces. Wiley, New York,
Conclusions pp 105–144
16. Pringle JH, Fletcher M (1983) Influence of substra-
At our studies we were demonstrated that
tum wettability on attachment of freshwater bacteria
wounds with critical colonization and proba- to solid surfaces. Appl Environ Microbiol 45:811–817
bly biofilms were faster healing if we were 17. Bendinger B, Rijnaarts HHM, Altendorf K, Zehnder
using dressings with antiseptics. Bacteria AJB (1993) Physicochemical cell surface and adhe-
18 T.P. Ručigaj

sive properties of coryneform bacteria related to the 34. Li X, Kong H, Mout R, Saha K, Moyano DF, Robinson
presence and chain length of mycolic acids. Appl SM, Rana S, Zhang X, Riley MA, Rotello VM (2014)
Environ Microbiol 59:3973–3977 Rapid identification of bacterial biofilms and biofilm
18. Williams V, Fletcher M (1996) Pseudomonas fluore- wound models using a multichannel nanosensor. ACS
scens adhesion and transport through porous media Nano 8:12014–12019
are affected by lipopolysaccharide composition. Appl 35. Nistico L, Gieseke A, Stoodley P, Hall-Stoodley L,
Environ Microbiol 62:1004 Kerschner JE, Ehrlich GD (2009) Fluorescence “in
19. Fletcher M (1988) The applications of interfer- situ” hybridization for the detection of biofilm in
ence reflection microscopy to the study of bacterial the middle ear and upper respiratory tract mucosa.
adhesion to solid surfaces. In: Houghton DR, Smith Methods Mol Biol 493:191–213
RN, Eggins HOW (eds) Biodeterioration 7. Elsevier 36. Oates A, Bowling FL, Boultin AJM, Bowler PG,
Applied Science, London, pp 31–35 Metcalf DG, McBain AJ (2014) The visualization of
20. Becker P, Hufnagle W, Peters G, Herrmann M (2001) biofilms in chronic diabetic foot wounds using rou-
Detection of different gene expression in biofilm-form- tine diagnostic microscopy methods. J Diabetes Res
ing versus planktonic populations of Staphylococcus 2014:153586
aureus using micro-­representational-­difference analy- 37. Malic S, Hill KE, Hayes A, Percival SL, Thomas DW,
sis. Appl Environ Microbiol 67:2958–2965 Williams DW (2009) Detection and identifi cation
21. Skrlin J (2016) Impact of biofilm on healing and a of specific bacteria in wound biofilms using peptide
method for identifying it in the wound. Acta Med nucleic acid fluorescent in situ hybridization (PNA
Croatica 70:29–32 FISH). Microbiology 155(Pt 8):2603–2611
22. Wolcott R (2014) Understanding biofilm formation 38. Davies D (2003) Understanding biofilm resis-
and biofilm-based wound care. Wound Middle East tance to antibacterial agents. Nat Rev Drug Discov
(Wound International) 1:24–26 2(2):114–122
23. Metcalf DG, Bowler PG, Hurlow J (2014) A clinical 39. Pulcini E (2001) The effects of initial adhesion events
algorithm for wound biofilm identification. J Wound on the physiology of Pseudomonas aeruginosa [Ph.D.
Care 23(3):137–138. 140-2 dissertation]. Montana State University, Bozeman
24. Kučišec-Tepeš N (2016) The role of antiseptics and 40. Bashan Y, Levanony H (1988) Active attachment
strategy of biofilm removal in chronic wound. Acta of Azospirillum brasilense Cd to quartz sand and
Med Croatica 70:33–42 to a light-textured soil by protein bridging. J Gen
25. Lewis K (2001) Riddle of biofilm resistance. Microbiol 134:2269–2279
Antimicrob Agents Chemother 45:999–1007 41. Danielsson A, Norkrans B, Bjornsson A (1977) On bac-
26. Lyczak JB, Cannon CL, Pier GB (2000) Establishment terial adhesion - the effect of certain enzymes on adhered
of Pseudomonas aeruginosa infection: Lessons from a cells in a marine Pseudomonas sp. Bot Mar 20:13–17
versatile opportunist. Microbes Infect 2:1051–1060 42. Nickel JC, Ruseska I, Wright JB, Costerton JW
27. James GA, Swogger E, Wolcott R, Pulcini E, Secor P, (1985) Tobramycin resistance of Pseudomonas aeru-
Sestrich J, Costerton JW, Stewart PS (2008) Biofilms ginosa cells growing as a biofilm on urinary catheter
in chronic wounds. Wound Repair Regen 16(1):37–44 material. Antimicrob Agents Chemother 27:619–624
28. James GA, Beaudette L, Costerton JW (1995) 43. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick
Interspecies bacterial interactions in biofilms. J Ind JS (2002) Extracellular DNA required for bacterial
Microbiol 15:257–262 biofilm formation. Science 295:1487
29. Gilbert P, Evans DJ, Brown MRW (1993) Formation 44. Billings N, Millan M, Caldara M, Rusconi R, Tarasova
and dispersal of bacterial biofilms in vivo and in situ. Y, Stocker R, Ribbeck K (2013) The extracellular
J Appl Bacteriol 74 Suppl:67S–78S matrix Component Psl provides fast-acting antibiotic
30. Marinović Kulišić S, Lipozenčić J, Tunuković S (2016) defense in Pseudomonas aeruginosa biofilms. PLoS
Antimicrobial dressings for infected ulcer and clinical Pathog 9:e1003526
comprehension of biofilm. Acta Med Croatica 70:23–27 45. Allesen-Holm M, Barken KB, Yang L, Klausen M,
31. Davies CE, Hill KE, Wilson MJ, Stephens P, Hill Webb JS, Kjelleberg S, Molin S, Givskov M, Tolker-­
CM, Harding KG, Thomas DW (2004) Use of 16S Nielsen T (2006) A characterization of DNA release
ribosomal DNA PCR and denaturing gradient gel in Pseudomonas aeruginosa cultures and biofilms.
electrophoresis for analysis of the microfloras of heal- Mol Microbiol 59:1114–1128
ing and nonhealing chronic venous leg ulcers. J Clin 46. Chiang WC, Nilsson M, Jensen PŘ, Hřiby N,
Microbiol 42(8):3549–3557 Nielsen TE, Givskov M, Tolker-Nielsen T (2013)
32. Kirketerp-Moller K, Jensen PO, Fazli M, Madsen Extracellular DNA shields against aminoglycosides
KG, Pedersen J, Moser C, Tolker-Nielsen T, Hoiby N, in Pseudomonas aeruginosa biofilms. Antimicrob
Givskov M, Bjarnsholt T (2008) Distribution, orga- Agents Chemother 57(5):2352–2361
nization, and ecology of bacteria in chronic wounds. 47. Mulcahy H, Charron-Mazenod L, Lewenza S (2008)
J Clin Microbiol 46(8):2717–2722 Extracellular DNA chelates cations and induces anti-
33. Davis SC, Ricotti C, Cazzaniga A, Welsh E, Eaglstein biotic resistance in Pseudomonas aeruginosa biofilms.
WH, Mertz PM (2008) Microscopic and physiologic PLoS Pathog 4:e1000213
evidence for biofilm-associated wound colonization 48. Walker TS, Tomlin KL, Worthen GS, Poch KR,
in vivo. Wound Repair Regen 182:6482–6489 Lieber JG, Saavedra MT, Fessler MB, Malcolm KC,
Biofilm: Clinical Experience 19

Vasil ML, Nick JA (2005) Enhanced Pseudomonas frequency ultrasound for patients with lower leg
aeruginosa biofilm development mediated by human ulcers due to chronic venous insufficiency: a report of
neutrophils. Infect Immun 73:3693–3701 two cases. Ostomy Wound Manage 60:52–61
49. Purdy Drew KR, Sanders LK, Culumber ZW, Zribi 64. Phillip PL, Yang Q, Schultz GS (2013) The effect of
O, Wong GC (2009) Cationic amphiphiles increase negative pressure wound therapy with periodic instil-
activity of aminoglycoside antibiotic tobramycin in lation using antimicrobial solutions on Pseudomonas
the presence of airway polyelectrolytes. J Am Chem aeruginosa biofilm on porcine skin explants. Int
Soc 131:486–493 Wound J 10(1):48–55
50. Kučišec-Tepeš N (2015) Antiseptics in the prevention 65. Gabriel A, Shores J, Bernstein B, de Leon J,
of chronic wound infection-Facts and misconcep- Kamepalli R, Wolvos T, Baharestani MM, Gupta S
tions. Acta Med Croatica 69(Suppl 1):91–99 (2009) A clinical review of infected wound treatment
51. Bjarnsholt T, Kirketerp-Mřller K, Kristiansen S, with Vacuum Assisted Closure (V.A.C.) therapy:
Phipps R, Nielsen AK, Jensen PŘ, Hřiby N, Givskov experience and case series. Int Wound J 6(2):1–25
M (2007) Silver against Pseudomonas aeruginosa bio- 66. Ichioka S, Watanabe H, Sekiya N, Shibata M,
films. APMIS 115(8):921 Nakatsuka T (2008) A technique to visualize wound
52. Castellano JJ, Shafii SM, Ko F, Donate G, Wright TE, bed microcirculation and the acute effect of negative
Mannari RJ, Payne WG, Smith DJ, Robson MC (2007) pressure. Wound Repair Regen 16:460–465
Comparative evaluation of silver-­containing antimi- 67. Besaer E, Kroukamp G, Wolfaardt GM, Boonzaaier
crobial dressings and drugs. Int Wound J 4:114–122 L, Liss SN (2010) Metabolic differentiation in bio-
53. Percival SL, Suleman L (2015) Slough and biofi lm: films as indicated by carbon dioxide production rates.
removal of barriers to wound healing by desloughing. Appl Environ Microbiol 76:1189–1197
J Wound Care 24:498–510 68. Hurlow J, Cough K, Laforet K, Bolton L, Metcalf
54. Heggers J, Goodheart RE, Washington J, McCoy L, D, Bowler P (2015) Clinical biofilms: A challenging
Carino E, Dang T, Edgar P, Maness C, Chinkes D (2005) frontier in wound care advances. Wound Care Adv
Therapeutic efficacy of three silver dressings in an (New Rochelle) 5:295–301
infected animal model. J Burn Care Rehabil 26:53–56 69. Attinger CH, Wolcott R (2012) Clinically address-
55. Percival SL, Bowler PG, Woods EJ (2008) Assessing ing biofi lm in chronic wounds. Adv Wound Care
the effect of an antimicrobial wound dressing on bio- (3):127–132
films. Wound Repair Regen 16:52–57 70. Ručigaj TP, Mihelic M (2015) Comparative effects of
56. Qian Z, Stoodley P, Pitt WG (1996) Effect of low-­ honey based and silver/charcoal based dressings on
intensity ultrasound upon biofilm structure from the healing of venous leg ulcers. Acta Med Croatica
confocal scanning laser microscopy observation. 69(1):67–72
Biomaterials 17:1975–1980 71. Planinšek Ručigaj T (2005) Venous leg ulcers
57. Qian Z, Sagers RD (1999) Pitt WG. Investigation of treatment with different alginate dressings (case
the mechanism of the bioacoustic effect. J Biomed study). In: Wundheilung Journal of Wound Healing
Mater Res 44:198–205 2, Sonderheft 2005; 9. Kongress der DGfW
58. Karau MJ, Piper KE, Steckelberg JM, Kavros SJ, Europäischer Wundkongress/European wound con-
Patel R (2010) In vitro activity of the Qoustic Wound ference, Stuttgart, 15–17
Therapy System against planktonic and biofilm bacte- 72. Košiček, M, TP Ručigaj. A comparative clinical trial:
ria. Adv Skin Wound Care 23:316–320 microbial colonisation of venous leg ulcers treated
59. Dong Y, Chen S, Wang Z, Peng N, Yu J (2013) Synergy with hydrofibre dressing or with ointments. In: 2nd
of ultrasound microbubbles and vancomycin against world union of wound healing societies meeting,
Staphylococcus epidermidis biofilm. J Antimicrob Paris, 8–13 July 2004
Chemother 68:816–826 73. TP Ručigaj, Somrak J Wound management with
60. Nishikori T, Ochi M, Uchio Y, Maniwa S, Kataoka H, foam with PHMB. In: EWMA meeting, Geneva,
Kawasaki K, Katsube K, Kuriwaka M (2002) Effects Switzerland, 26–28 May 2010
of low-intensity pulsed ultrasound on proliferation 74. TP Ručigaj, Kecelj N, Slana A Naše izkušnje s prvo
and chondroitin sulfate synthesis of cultured chondro- slovensko oblogo za zdravljenje kroničnih ran pri
cytes embedded in Atelocollagen gel. J Biomed Mater inficiranih venskih golenjih razjedah: kontrolirana
Res 59:201–206 študija. In: Advanced treatment of chronic wounds
61. Nishikawa T, Yoshida A, Khanal A, Habu M, and infected tissue/6th symposium on wounds,
Yoshioka I, Toyoshima K, Takehara T, Nishihara Portoroz, 12–13 May 2011
T, Tachibana K, Tominaga K (2010) A study of the 75. Planinšek Ručigaj T (2006) Treatment of venous leg
efficacy of ultrasonic waves in removing biofilms. ulcers with different alginate dressings: their effects
Gerodontology 27:199–206 on healing rate and pain: randomized clinical study:
62. Escandon J, Vivas AC, Perez R, Kirsner R, Davis S poster 113. V: Innovation, education, implementation:
(2012) A prospective pilot study of ultrasound ther- final programme, abstracts. Prague: European Wound
apy effectiveness in refractory venous leg ulcers. Int Managemnet Association, 2006, str. 154
Wound J 9:570–578 76. Rucigaj TP* (2005) [posterabstract] 159. Zeitschrift
63. Maher SF, Halverson J, Misiewicz R, Reckling T, für Wundheilung, 2005, sonderheft 2, str. 276.
Smart O, Benton C, Schoenherr D (2014) Low-­ [COBISS.SI-ID 22411832]
 Mixed-Species Biofilm
Compromises Wound Healing
by Disrupting Epidermal Barrier
Function

Terri A. Zomerlei and Gayle M. Gordillo

1 Introduction t­issue remains in a prolonged and persistent

inflammatory state. However, the mechanisms of
In the United States alone, over 6.5 million delayed wound healing secondary to biofilm
patients are plagued with chronic wounds [1]. presence remains poorly understood.
The financial burden of chronic wounds on the For the clinician, understanding of chronic
healthcare system is colossal with conservative wound biofilm biology has been limited by mul-
estimates approaching an annual cost of $25 bil- tiple challenges. First, it is difficult to reliably
lion dollars [2]. More recent research has revealed culture biofilm-producing bacteria. The diagno-
that the source of many chronic wounds is likely sis of biofilm infection is challenging as there are
attributable to biofilm. Biofilm is a specific no reliable testing modalities that are readily
arrangement of bacteria in which they form an available in the clinical setting [4]. In addition,
organized and cohesive community. In the bio- in vivo animal models have not allowed for eval-
film form as opposed to the free-floating plank- uation of the longitudinal growth and mecha-
tonic state, bacteria are encased within a durable nisms of pathogenesis of biofilm, thus limiting
matrix called extracellular polymeric substance the translation of bench research results to the
(EPS). The Centers for Disease Control and the clinical understanding of chronic wounds [5–7].
National Institute of Health estimate that 60–80% Furthermore, the biology of biofilm pathogenesis
of chronic wounds can be attributed to biofilm is determined not only by the relations of the bac-
colonization [3]. teria within the biofilm, but also with the bacteria-­
The primary role of skin is to act as a barrier to host interactions. The bacteria-host interactions
the external environment. When an insult occurs are intricate and play a critical role in defining
to the skin resulting in a wound, the healing pro- human disease conditions, which limits the clini-
cess begins. The presence of biofilm in chronic cal validity of in vitro studies.
wounds has been shown to delay wound healing. Given these challenges, Roy et al. [6] devel-
There are two common features of wound biofilm oped a mixed-species biofilm infection that satis-
infection: they render the bacteria almost imper- fied many necessary criteria in order to
vious to antimicrobial agents and the affected appropriately translate research conclusions. The
model utilizes a porcine host, several biofilm-­
producing bacteria, a wound that is of full
T.A. Zomerlei • G.M. Gordillo, M.D. (*) ­thickness, and wound infection that is sustained/
Department of Plastic Surgery, Ohio State University chronic [2, 6]. As described previously, character-
Wexner Medical Center, 915 Olentangy River Road,
Suite 2100, Columbus, OH 43212, USA
istics of biofilm include multiple studied mecha-
e-mail: [email protected] nisms that contribute to wound chronicity

Recent Clinical Techniques, Results, and Research in Wounds (2017) 21

DOI 10.1007/15695_2017_3, © Springer International Publishing AG
Published Online: 24 October 2017
22 T.A. Zomerlei and G.M. Gordillo

including the ability to resist antimicrobials, evade Therefore in order to confirm a biofilm infection
host immune defenses, and disable skin barrier with this model and meet the established crite-
function of the host. While the biofilm’s ability to rion several additional tests were performed.
resist antimicrobials and evade host defenses has To ascertain adherence to a surface within an
been well established, the effect of biofilm on the EPS substrate, wound biopsies were obtained
function of skin has been more recently elucidated 7 days post-inoculation and examined with scan-
by the work of Roy et al. [6]. The following ning electron microscopy (SEM). This revealed
“Technique” section outlines the methods and the presence of both organisms within EPS in the
results obtained by this research group. burn wounds. In addition, a scrubbing technique
was utilized to remove planktonic bacteria from
the wounds. Microbiologic analysis revealed that
2 Technique the bacterial counts did not substantially change
following the scrubbing, indicating that the bac-
In order to study the effect of biofilm on chronic teria in a biofilm state were tightly adherent to the
wounds, a large animal porcine model of biofilm soft-tissue bed of the wound. To confirm a persis-
infection was developed. A clinically relevant tent and localized infection, wound biopsies were
mixed-bacterial species infection that combined taken on days 14 and 35 post-inoculation that
Acinetobacter baumannii and Pseudomonas also confirmed biofilm presence. Localized infec-
aeruginosa was established [8, 9]. In addition, a tion was verified with negative blood cultures and
heated press that administered uniform tempera- lack of systemic signs of infection. The ability of
ture, pressure, and duration of thermal energy the biofilm to resist treatment with antibiotics
transfer to create a full-thickness burn wound of was substantiated by treating the wound with
uniform consistency was created. Three days Acticoat 7™, as silver dressings are the standard
after the burn wounds were made, the wounds of care for management of infected burn wounds
were either inoculated with Acinetobacter bau- [12]. The planktonic forms of A. Baumannii and
mannii and Pseudomonas aeruginosa or left P. aeruginosa were effectively eradicated with
untreated to serve as spontaneously infected, i.e., Acticoat 7™. However, treatment of the chronic
whatever contaminated the wounds from the burn wounds with the silver dressing was ineffec-
environment, controls. The burn wounds were tive against the bacteria in the biofilm state.
then examined at 7 days post-inoculation. To Additional investigations that verified the pres-
confirm that a biofilm infection exists, several ence of a biofilm in this model included recur-
qualifications need to be met. These criteria rence after debridement and induction of
include adherence of infecting bacteria to a sur- biofilm-specific genes in the Acinetobacter bau-
face or each other [10], visual evidence that the mannii and Pseudomonas aeruginosa [13].
bacteria are encased in EPS [9], persistent and Once the wound biofilm model was confirmed
localized infection [4], and demonstration of to be sound, the interactions of the biofilm with
antibiotic resistance despite susceptibility of that the host were studied longitudinally. Currently,
bacteria in the planktonic state (Table 1) [11]. wounds are considered “healed” when wound
closure is obtained and confirmed by visual
inspection. In regard to closure based on visual
Table 1 The four criteria proposed by Parsek and Singh
to define biofilm infection [11] observance, the control as well as the biofilm-­
infected wounds were comparable. However, to
1 Adherence of infecting bacteria to a surface
verify that the healed skin has reestablished its
2 Direct visual evidence that bacteria are encased in
EPS barrier function, a transepidermal water loss
3 Confinement of the infection to a particular location (TEWL) measurement was obtained. The mea-
4 Demonstration of antibiotic recalcitrance despite surements are obtained noninvasively using a
known susceptibility of the bacteria in the probe (Fig. 1). A higher TEWL measurement
planktonic state indicates that the barrier function of the skin is
Mixed-Species Biofilm Compromises Wound Healing by Disrupting Epidermal Barrier Function 23

f­unction are zona occludens-1 (ZO-1) and zona

occludens-2 (ZO-2). These were found to be sig-
nificantly downregulated in biofilm-infected burn
wounds. In addition, two micro-RNAs (miR-­
146a and miR-106b) were significantly induced
in keratinocytes in the biofilm-infected wounds
compared to controls. To establish a relationship
to these two findings, an in vitro model of kerati-
nocytes exposed to mixed-species biofilm was
studied. The in vitro model supported the previ-
ous in vivo findings and ZO-1 and ZO-2 were
silenced while miR-146a and miR-106b were
upregulated. Additionally, P. aeruginosa, a com-
ponent of the mixed-bacterial biofilm model,
only upregulated the key miRs in the biofilm phe-
Fig. 1 Transepidermal water loss measurement.
DermaLab TEWL Probe (cyberDERM Inc., Broomall, notype and not in the planktonic state. In order to
PA) was used to measure transepidermal water loss connect the miRs to ZO-1 and ZO-2 directly,
expressed in gm/m2/h immunocytochemical studies were performed
which revealed that the miRs expression was
impaired. Therefore, TEWL measurement in inversely associated with the presence of corre-
addition to visual inspection was performed to sponding ZO proteins. A final confirmatory study
objectively and quantitatively determine the rees- involved placing miRNA mimics in a topical
tablishment of the skin barrier function in the cream-based delivery system and applying it to
burn wounds. Intriguingly, although the visual mouse skin. Analysis of the mouse skin revealed
skin assessment of a healed wound infected with that the key proteins ZO-1 and ZO-2 were
biofilm appeared similar to the burn wound with- silenced and in turn the barrier function of the
out biofilm infection, the barrier function of the skin was compromised as manifested by
skin was markedly impaired in the wounds increased TEWL [6].
infected with biofilm compared to the controls.
The biofilm-infected TEWL measurements
peaked at 14 days and remained consistently ele- 3 Discussion
vated at that level even out to post-inoculation
day 56. The burn wounds without biofilm infec- Biofilm presents a diagnostic and therapeutic
tion had elevated TEWL measurements that also challenge to clinicians. Biofilm has become
peaked at 14 days, but then progressively seemingly ubiquitous in those afflicted with
decreased and returned to levels observed in nor- chronic wounds and the ramifications of biofilm
mal unburned skin by 56 days post-inoculation. infections plague the health systems. Biofilm-­
Thus, despite the appearance of wound closure producing bacteria are able to act synergistically
with intact skin, the biofilm-infected wound had to aid in collective survival, thus further compli-
impaired skin that left the wound functionally cating clinical treatments [14–17]. Not only are
open [6]. these biofilm communities resistant to healthy
The barrier function of the skin is dictated by host innate immune responses, but also their
the integrity of the adhesive interactions at the unyielding and protected adherence to surfaces
epithelial apical junction complex. These adhe- results in difficulty identifying the bacteria by
sion interactions are primarily tight junctions, standard culture methods [3, 18]. A 2013 study of
adherens junctions, desmosomes, and gap junc- deep sternal wound infections (SWI) revealed
tion proteins. Two central tight-junction proteins that although scanning electron microscopy
that are crucial in maintaining skin barrier ­analysis from six study patients revealed dense
Another random document with
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 “I don’t know, I’m sure; we never thought of it,” replied Miss
Ferrars meekly.
“If you had it would not have done you much good,” put in
Geoffrey; “there are no carriage-horses. I never knew such a little
duffer as Alice—sending them back to Looton,” he added in a low
aside.
“No carriage-horses!” echoed Mrs. Blundell, whose ears had at
least caught that sentence. “Dear me! you don’t say so?” in a tone of
deep commiseration. Then turning aside to her friend she whispered
(?): “I heard he kept her tight, but I had no idea it was as bad as
that.”
Mary, Geoffrey, and Mr. Mayhew exchanged looks of unqualified
amazement, and again an awkward silence ensued.
Mrs. Blundell once more proceeded in a louder and more forte
key:
“I am surprised to see Lady Fairfax entertaining visitors; I had no
idea she ever had people staying here.”
“We are the exception that proves the rule,” replied Geoffrey at the
top of his naturally robust organ.
“Are you staying in the house—you two young men?” indicating
Geoffrey and Reginald with a fat forefinger.
“Yes,” returned Geoffrey, who had taken upon himself the task of
answering.
“Ah! I do not think I know your face,” to Geoffrey. “Are you in the
Manister Bank?” patronisingly.
“No, I’m not;” rather sharply.
“Do you belong to this part of the country?”
“I have not that honour.”
Mrs. Blundell gazed at him dreamily for nearly sixty seconds, and
then a light seemed to break, for she exclaimed with the triumph of
one who has grasped and presents an indisputable fact:
 “I have it! You are the new young man in the Brewery.”
“I am not,” returned Geoffrey haughtily, and shouting with
impressive distinctness. “I am not in the Brewery; and to save you
the trouble of further speculation on my behalf, I may as well inform
you that I’m in the cavalry.”
“Ah!” There was a world of meaning in that interjection—a
meaning no pen could convey. “And he?” indicating Reginald with
her sunshade.
“Cavalry officer also.”
“Two cavalry officers,” she repeated slowly, evidently rehearsing
the intelligence for future occasions. If she had said, “Two returned
convicts,” her intonation could not have expressed deeper
disapproval.
Whilst she was gratifying her thirst for information, her friend and
Mrs. Mayhew were exchanging platitudes about flowers and fruit, the
seasons of the year, and suchlike enthralling topics. They now made
a combined effort to include her in their conversation. But it was of
no avail; she evidently preferred to draw out Geoffrey, who seemed
not merely willing, but delighted to oblige her.
Having replenished her cup with politest alacrity, he resumed his
seat in front of her à la Turc, and looked up at her with an amused
twinkle in his mischievous little hazel eyes.
“Lady Fairfax is a very pretty young woman,” she remarked to him
over her teacup. A nod satisfied her of Geoffrey’s cordial assent. “My
son admires her immensely, so do all the gentlemen about here. She
is rather what I call a gentleman’s beauty,” she added in a
deprecating tone; “but still I think her decidedly good-looking,” with
an air that signified that Alice had now, and once for all, received an
invaluable cachet of distinction.
“Very kind of you, I’m sure,” muttered Geoffrey.
“Frederick has been most anxious for me to call ever since he met
Lady Fairfax one day out riding; he has been dying to make her
acquaintance. He has such an eye for beauty.”
 “He looks like it,” assented Geoffrey in a cheerful shout.
“Be quiet, Geoffrey,” muttered Reginald from behind.
“Are you any relation to Lady Fairfax, may I ask?”
“Yes, of course I am,” roared Geoffrey.
“Both of you? Cousins did you say?”
“No, I did not; but I am her cousin.”
“As much her cousin as I am,” in a loud aside to her friend, and
with a significance baffling all description.
Mary, seeing a storm brewing in Reginald’s eyes, hastened to
throw herself into the breach with an all-absorbing bazaar for bait.
But no, the devoted old lady madly rushed on her fate. After a few
brief replies she resumed:
“Did you say that this other gentleman was a cousin also?”
regarding Geoffrey with a keen satirical eye.
“The interest you take in Lady Fairfax is most gratifying to the
whole family. No, he is not her cousin, he is her husband.”
“Not her cousin, not her husband! You need not tell me that; of
course I know that,” with insolent emphasis.
What was to be done with this terrible old woman, on whom her
friend’s signs and nudges were entirely thrown away?
At this instant, the game over, Alice, flushed and breathless, joined
the group.
“I won, Geoff; only—fancy—that,” she said, laying her hands on
his shoulders in the excitement of her recent victory.
“Then, I suppose, there will be no living in the same house with
you for the next week,” remarked her cousin, moving so as to make
room for her beside him on the grass.
She looked utterly fagged and exhausted; her frail delicate
appearance struck her husband forcibly, and for the first time he
sprang up, dragged forward a garden-chair, and, taking her by the
arm, pushed her into it with an air of loverlike solicitude—by no
means lost on Mrs. Blundell—that had been foreign to his manner for
many a long day.
“Thank you, Reginald,” said Alice, sinking back into the seat with a
sigh of relief and removing her hat. “To reward you for your
politeness you shall have a little bit of my dress to sit on,” spreading
out the folds of her skirt.
“This is really too barefaced,” cried Mrs. Blundell in one of her very
loudest asides.
Then, getting up and extending her hand very stiffly to Alice, she
said in a most pointed unmistakable manner:
“It is quite time for me to be going, Lady Fairfax. I wish you good-
afternoon. Come, Frederick,” she called to her son, who was
quaffing quantities of claret-cup, “I am ready,” and with a
comprehensive bow she was sailing off, but was arrested by Sir
Reginald, who, leaping to his feet, confronted her.
“Before you leave, madam, will you have the goodness to tell me
who you think I am?”
With a most evil and significant smile she was turning away, and
metaphorically proceeding to shake the dust off her feet, when he
again detained her.
“I am Lady Fairfax’s husband!” he shouted. “What do you mean by
your looks and innuendoes?”
“What is he saying, Frederick? I can’t hear a word.”
Reginald, turning to her son, with eyes ablaze and perfectly livid
with passion, said to the electrified youth: “Be good enough to make
your mother understand who I am; also make her clearly
comprehend that neither Lady Fairfax nor myself have any further
desire for her acquaintance. As for you”—with withering contempt
—“I sincerely hope your curiosity has been gratified with regard to
my wife’s appearance. That there may be no delay in your
departure”—looking at the three culprits sternly—“I shall myself go
and order your carriage.”
 So saying, he took off his hat and walked away, leaving his visitors
covered with amazement and confusion, Geoffrey in agonies of
repressed laughter, and Miss Ferrars and Mrs. Mayhew in a state of
mental coma.
When this tirade had been interpreted to Mrs. Blundell—she had
heard a good deal more than she pretended—she returned across
the grass, from where she was awaiting her carriage, and humbly
accosting Alice, overwhelmed her with excuses and apologies which
there was no avoiding. The worldly-wise old lady said to herself: “It
will never do to quarrel with the Fairfaxes—people of great wealth
and influence, if all is as it seems. Supposing her outrageous
mistake was to get about, what capital for her fellow-gossips! At all
costs she would leave on friendly terms, and be literally stone deaf to
every snub.” Summoning a sweet smile to her discomfited
countenance, she implored Alice to intercede with her husband: “He
looks as if he could refuse you nothing. Do make my peace with him;
do go and bring him to receive my most humble apologies. You must
blame my unfortunate deafness, not me. I am not like other people,
my dear young lady; I am afflicted, and I frequently get hold of wrong
impressions, which is my great misfortune—not, I am sure you will
allow, my fault. I did hear a little idle whisper that you were rather—a
—rather—a——” casting wildly about for a delicate way of
expressing herself, and becoming crimson in the attempt—“shall we
say—fast young lady?”
“Certainly, if you like; and as long as I need not agree to the fact,”
returned Alice with much composure.
“Well, and finding you entertaining three cavalry officers, all on a
most familiar footing, and imagining that your husband was still
absent, I just thought, as a much older married woman”—effusively
—“I would give you a little hint by my manner.”
“In that she succeeded to a marvel,” murmured Geoffrey.
“And I had no idea, no more than the man in the moon, of the real
state of the case; nor that that dark distinguished-looking young man
was Sir Reginald himself. And has he come to stay? and where has
he been all this time?” she asked with affectionate solicitude.
“However, I’ll question you another time. Do run after him and obtain
my forgiveness; I assure you I cannot leave the place without it,”
planting her parasol in a typical manner in the sod and waving Alice
to the quest.
Alice most unwillingly set out to find her husband; he was in the
yard composing himself with a cigar, and personally despatching the
carriage. When he had heard what she had to say he burst forth:
“Alice, I am astonished that you can ask such a thing. No, I
certainly will not forgive them; and if you say another word on the
subject, I warn you that I shall begin to swear. I feel literally boiling
with rage. Nothing less than a swim in the river will cool me,” he
observed, moving off.
“Stay one instant,” she cried, running after him. “What am I to say
to them, then?”
“Say? Oh say that I am in such a frightful rage you are afraid to go
near me.”
“But you are not quite so bad as all that, and I am not the least
afraid of you,” she returned with a smile.
“Are you not?” he said, taking his cheroot out of his mouth and
looking hard at her. “Well, you may go back and tell them that I
forgive them this time for your sake, since you say that nothing else
will induce the old woman to quit the premises.”
“You will not come back and say so yourself?” she asked
insinuatingly.
“Not for ten thousand pounds; my forgiveness is but hollow. I
should like nothing better than to give that young booby a thrashing
that would surprise him, and to duck his mother in the pond. Such
are my savage instincts. That is what I would do if I were a North
American Indian and you were my squaw,” he concluded with a grim
smile.
“Reginald, I think you have taken leave of your senses.”
“I see one thing very plainly,” he continued, walking by her side to
the edge of the lawn, “and that is, that I shall have to stay here much
longer than I intended, to rehabilitate you in the good opinion of
society. So be prepared to enact with me in public the part of a most
united happy couple. Do you understand?” he said, throwing the end
of his cigar among the laurel bushes and coming to a full stop. “I will
accompany you everywhere, carry your fans, shawls, bouquets, and
other loose paraphernalia, and you”—very bitterly—“must assume a
certain amount of interest and gratitude in return for my devoted
solicitude. It will only be for a short time, but I see that it is an
imperative though disagreeable necessity.”
So saying, he turned abruptly away down a side walk, leaving
Alice with tears of mortification smarting in her eyes.
 CHAPTER X.
GEOFFREY MANŒUVRES.

An hour later Reginald made his appearance in the library, where

he found all the party assembled except Alice. Seeing him look
round the room, Helen volunteered to tell him that she had gone to
see a sick girl.
“What, at this time of night?”
“She went nearly an hour ago. She insisted on going, as she had
not been to see Lucy Summers for some days. Alice has been so
good to her all the summer—she is dying of consumption, poor girl.”
“It is quite time that Alice was home,” said her husband with
authority. “Half-past seven!” walking to the window and looking at his
watch.
“Geoffrey promised to fetch her. You ought to start, Geoff,” said
Helen. “You know that this is market-night, and her abject fear of
drunken men is no secret.”
“She need not go as far as the road for them,” remarked Reginald.
“Just now I met an under-gardener endeavouring to walk up both
sides of the avenue at once.”
“Come, Geoff, you had better be off if you are going.”
“Oh, I’m exhausted,” replied Geoffrey. “I really could not think of
taking any more exercise to-day.”
“But you promised,” urged his cousin emphatically.
“Promised, did I?” he replied, rising languidly and deliberately
arranging a cushion behind his head as he settled himself into the
snuggest corner of the sofa. “Oh, Alice is accustomed to my
promises by this time; she knows they are like piecrust—made to be
broken. Besides, Alice has a young and active husband. Pedestrian
exercise is good for these Anglo-Indians; let him go.”
“But, Geoffrey——”
“‘But me no buts;’ I won’t stir till the first bell rings, if then. That girl
has already run me off my legs, and if she is mad enough to start for
a two-miles’ walk at this time of night, I am not. I prefer lying here”—
shutting his eyes—“and thinking of dinner.”
“Well, Geoffrey,” exclaimed Reginald indignantly, taking up his hat,
“if you won’t go, I must. Where does this sick girl live?”
“Go out by the lower avenue, turn to the left, and follow your nose
—it’s straight, isn’t it?—till you come to a plantation; go through that,
and you will see a field, and in the field a cottage. And you had better
look sharp, my dear boy; it’s getting late.”
As the door closed, Geoffrey started up and began capering about
the room.
“Did I not do that splendidly?” he asked, stopping and rubbing his
hands. “Haven’t I arranged for a nice little conjugal tête-à-tête, and
isn’t he just swearing at me! Ten to one they will have a battle-royal,
but anything is better than this armed peace; the way in which they
avoid each other is a most beautiful study in tactics.”
“If you will take my advice,” observed Mrs. Mayhew, “you will not
put your finger in the pie. Leave it to time, and it will all come right.”
“I don’t agree with you there,” replied Geoffrey. “Leave all to time
and it will all go wrong, unless time is assisted by kind friends who
make such judicious arrangements as this walk for example. They
require as much looking after as if it were a half-developed love
affair.”
“Why should you busy yourself about them, an unfledged
youngster like you?” asked the Honorable Mark peevishly.
Perfectly ignoring the question, Geoffrey stalked over to Helen,
delightfully unconscious that an antimacassar was clinging to his
coat-tails.
 “Helen, now that we are here, ‘en champ clos’—or to translate it
freely, Miss Ferrars and auntie are gone to dress, and the master
and mistress are out—tell me honestly what you think about the
business—will it all come right, or will he hook it off to the wars
again?”
“What a way of expressing yourself! What polished ease! Well, if
you want my opinion, you are quite welcome to it. I think the
prospect is decidedly gloomy.”
“You do? Well, listen to me—I am certain that his cool indifference
is only assumed—is that nicely expressed?—and, as to her, I
daresay she is quite ready to kiss and be friends. Suppose you
break the ice with her, and I’ll put out a feeler in his direction?”
“Helen,” almost shouted her husband, “don’t attempt to interfere,
whatever Geoffrey may do—and he has assurance for twenty. But
you’ll see he will only burn his fingers,” added Mr. Mayhew
emphatically.
“Never mind him, Helen, you back me up,” urged Geoffrey eagerly.
Helen merely shook her head in reply.
“Pouff! Mr. Mayhew,” he expostulated indignantly, “I had a much
better opinion of you. You have no pluck!”
So saying, he lounged out of the room, banging the door loudly
after him.
 CHAPTER XI.
“MEET ME BY MOONLIGHT ALONE.”

In the meantime, Sir Reginald was walking rapidly in the direction

of the Summers’ cottage. He reached the wood, which was thickly
planted, and covered about an acre of ground. Spruce and fir made
it dusky even in the daytime, and now in the twilight it was almost
pitch-dark. Vaulting over the stile, he followed a path till he came to
another stile, near which was the cottage, as Geoffrey had
described.
“I’ve come far enough,” he said to himself, “and whilst I wait I’ll
have a smoke.” So, leaning against a tree, he struck a light, and lit at
least his sixth cigar that day. After five minutes or so he saw the
cottage door open, and a white dog and a slender white figure
emerge, both of which started off at a brisk run across the field, Alice
collapsing to a sober walk as she neared the plantation. Stepping
lightly over the stile, she advanced cautiously through the gloom, but
descrying the spark at the end of her husband’s cheroot, she
exclaimed, as she sprang towards him and seized his arm:
“Oh, Geoff! you good boy, I was half afraid you would not come. I
never was more glad to see you—I do so hate this lonely dark wood.
They say a murder was committed here years ago,” she added,
drawing closer to him and shuddering. “Come, we must be quick,”
she chattered on; “I shall get into dreadful hot water, I am so late,
and I am so tired I can hardly crawl. Not that I mind, only Helen
makes such a fuss if she sees me looking pale and sleepy. Why
don’t you speak, you lazy fellow? you are always smoking. Who
would think you had such an arm,” pinching him; “it’s like a
blacksmith’s; the muscles feel as if they would burst the sleeve of
your coat. I shall have no compunction in leaning pretty heavily, I can
tell you.”
 “Are you dumb, Geoffrey; or are you in the sulks?”
A sudden idea struck her. It was not Geoffrey after all; perhaps—
agonising thought!—it was some utter stranger whom she had thus
cavalierly appropriated.
“What have I done?” she cried, horror-struck, and endeavouring to
release her hand. “Please let me go, whoever you are,” she pleaded
piteously.
By this time they were close to the road, and by the light of the
newly-risen moon she saw her husband, and stood aghast.
“Geoffrey was, or said he was, too lazy to come,” he remarked,
helping her over the stile, “so I came as his substitute. I daresay you
will find my arm quite as efficient a support,” coolly replacing her
hand.
“Oh, but indeed,” struggling to withdraw it, and struggling in vain, “I
never dreamt it was you, or I would not—I would not——”
“Have taken such a liberty,” he interrupted. “No, I daresay not.”
“There is no necessity to show me such politeness now,” she
exclaimed hotly; “it is only in public, as you said yourself, that you
are to pay me any attention. Let my hand go, please; I can walk very
well without any assistance.”
“Nevertheless, as you admitted just now that you were tired, you
will have to do violence to your feelings for once and accept my arm,
much as you dislike it; and if the high road is not a public place, I
should like to know what is. Why did you not defer this visit till to-
morrow? No wonder you are tired, after playing lawn-tennis all the
afternoon. What can have possessed you to take such a walk?” he
asked, slackening his pace.
“I could not have slept,” she rejoined, “if I had not, for I had not
been to see Lucy for a week, and my conscience was telling me I
had neglected her.”
“Oh then you have a conscience?” he observed gravely.
“Of course I have. What an odd question! Why do you ask?”
 “Mere idle curiosity. Who is this Lucy Summers you have been to
see?”
“A girl who is very ill; she thinks so much of my visits, poor thing;
but she does me far more good than I have it in my power to do her.
She is truly fit for heaven, if anyone can be so.”
“She is dying, is she not?”
“Yes, of consumption; and she is only my age. If I were like her I
should be glad to go—only for Maurice.”
A long and truly eloquent silence, lasting for fully a quarter of a
mile. Alice thought of the last time they had walked together arm-in-
arm up and down the long gallery at Looton, the evening before he
had started for Cannes. What an age it seemed since then! What
changes had occurred! He was more changed than all else, she felt,
as she stole a glance at him. His clear-cut profile looked coldly
severe in the moonlight, his eyes were fixed on the horizon, and his
thoughts seemed at least a thousand miles away. The moon, which
had risen behind the park trees, was now sailing proudly overhead,
and looked down full-faced on this strangely-silent couple.
The rattle of an approaching dog-cart and the sound of a horse’s
hoofs aroused them from their reflections.
Two young men in evening dress, evidently going out to dinner.
They favoured Alice with a hard stare, and Reginald with a knowing
look, as they dashed past.
“Pretty girl!” and “Lucky dog!” was borne upon the breeze as they
rounded a corner, leaving behind them a cloud of dust.
As Alice put up her hand to ward off a volume of it, her wedding-
ring glittered in the moonlight, and, for the first time, caught her
husband’s eye.
“So you have replaced your wedding-ring, I see,” he observed, as
they entered the avenue-gates.
“I have,” she replied in a low voice.
 “What an interesting ceremony it must have been,” he remarked
sarcastically.
“What do you mean?” asked Alice, gazing up at him with
unrestrained astonishment.
“Did you swear to love, honour, and obey Alice Fairfax? I have
often heard of people being wedded to self, but such an utterly
barefaced proceeding as yours I never met with before.”
Alice had never thoroughly realised till now how bitterly he had
resented her treatment of his wedding-ring.
“Where is my own ring?” she asked with a reckless boldness that
surprised herself.
“I wear it on my watch-chain.”
“Will you ever give it back to me?” she inquired, more and more
amazed at her own audacity.
He paused and stood still for a moment, and eyeing his wife with
cool unspeakable amazement, said:
“Will I give you back your wedding-ring? When you deserve it I
may; but,” he added slowly and impressively, “as far as I can judge
at present, that will never be.”
He felt her little hand tremble on his arm, he saw her lips quiver, a
mist come over her deep-fringed eyes. Seized with sudden
compunction, he said:
“I am afraid I am always giving you rude brusque answers, but you
brought this on yourself. The past three years have not been
calculated to improve a man’s temper, have they?”
She looked up.
“You know you don’t deserve your wedding-ring, do you?” said he,
taking her hand. “Do you?” he added pertinaciously.
“I suppose not,” faltered Alice, gulping down her tears with a
painful effort.
 “You suppose not!” he echoed impatiently. “Well, I am very certain
you don’t; and the ring is likely to remain in my keeping.”
By this time they had reached the hall door-steps, where Geoffrey,
in full evening dress and the usual flower in his button-hole, was
awaiting them.
“At last!” he exclaimed. “So you have really come home. Well, you
did not hurry yourselves,” he said, escorting them into the hall. “We
began to think you had eloped—gone off together into some elegant
retirement in the style of a second honeymoon.”
“Geoffrey!” cried Alice, in an agony of blushes.
“Don’t ‘Geoffrey’ me, my good girl, but go and get ready for dinner
as quickly as you can; I’m starving.”

END OF VOL. II.

CHARLES DICKENS AND EVANS CRYSTAL PALACE PRESS.

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