Apologies, but just so it is not missed.

Biofilm bacteria: formation and comparative susceptibility to
antibiotics

Merle E. Olson, Howard Ceri, Douglas W. Morck, Andre G. Buret, and
Ronald R. Read
Biofilm Research Group (Olson, Ceri, Morck, Buret, Read), Microbiology
and Infectious Diseases (Olson, Ceri, Read), Department of Biological
Sciences (Olson, Ceri, Morck, Buret), University of Calgary, Calgary,
Alberta T2N 4N1.

Abstract
The Calgary Biofilm Device (CBD) was used to form bacterial biofilms
of selected veterinary gram-negative and gram-positive pathogenic
bacteria from cattle, sheep, pigs, chicken, and turkeys. The minimum
inhibitory concentration (MIC) and minimum biofilm eradication
concentration (MBEC) of ampicillin, ceftiofur, cloxacillin,
oxytetracycline, penicillin G, streptomycin, tetracycline,
enrofloxacin, erythromycin, gentamicin, tilmicosin, and trimethoprim-
sulfadoxine for gram-positive and -negative bacteria were determined.
Bacterial biofilms were readily formed on the CBD under selected
conditions. The biofilms consisted of microcolonies encased in
extracellular polysaccharide material. Biofilms composed of
Arcanobacterium (Actinomyces) pyogenes, Staphylococcus aureus,
Staphylococcus hyicus, Streptococcus agalactiae, Corynebacterium
renale, or Corynebacterium pseudotuberculosis were not killed by the
antibiotics tested but as planktonic bacteria they were sensitive at
low concentrations. Biofilm and planktonic Streptococcus dysgalactiae
and Streptococcus suis were sensitive to penicillin, ceftiofur,
cloxacillin, ampicillin, and oxytetracycline. Planktonic Escherichia
coli were sensitive to enrofloxacin, gentamicin, oxytetracycline and
trimethoprim/ sulfadoxine. Enrofloxacin and gentamicin were the most
effective antibiotics against E. coli growing as a biofilm. Salmonella
spp. and Pseudomonas aeruginosa isolates growing as planktonic
populations were sensitive to enrofloxacin, gentamicin, ampicillin,
oxytetracycline, and trimethoprim/sulfadoxine, but as a biofilm, these
bacteria were only sensitive to enrofloxacin. Planktonic and biofilm
Pasteurella multocida and Mannheimia haemolytica had similar
antibiotic sensitivity profiles and were sensitive to most of the
antibiotics tested. The CBD provides a valuable new technology that
can be used to select antibiotics that are able to kill bacteria
growing as biofilms.
Top
Abstract

Materials and methods
Results
Discussion
References

Introduction

Historically, we have studied these microorganisms by culturing them
in highly enriched liquid or solid media that artificially selects for
less hardy bacteria (1,2,3). However, bacteria exist within natural
systems in an entirely different form from these artificially grown
laboratory strains (1,2,3). In order for bacteria to survive within
hostile environments such as that encountered in host tissue
(antibodies, phagocytes, etc.) or on an inert surface exposed to
inhospitable conditions (UV light, desiccation, heat, cold, shear
forces), they have adapted by existing as adherent populations
(sessile bacteria). Sessile bacteria appear to be protected in these
antagonistic environments by growing as colonies encased in an
extracellular matrix of carbohydrate or exopolysaccharide (1,2,3,4). A
large collection of these groups of bacterial cells adhering to a
surface is called a bacterial biofilm (1,2,3). When bacteria are
examined in natural environments and within infected tissue, biofilms
are the most predominant form. Sessile bacteria growing on surfaces
have nutrient limitations and so may grow more slowly and have
restricted mobility (4); planktonic forms in culture media have
unnatural access to nutrients, multiply rapidly and often are highly
motile. Planktonic bacteria are more susceptible to the effects of
antibiotics and to environmental and host factors (1,2,3,4).
Conversely, sessile bacteria are able to resist or evade such
destructive factors by forming aggregates, altering their physiology,
and taking advantage of deficiencies in the host clearance mechanisms
(1,2,3,4).

Many common bacterial pathogens exist in animals as biofilms. Typical
animal diseases where bacterial biofilms are believed to be involved
based on histopathologic and ultrastructural appearance of the
bacteria within tissue include: mastitis (Streptococcus agalactiae,
Staphylococcus aureus), pneumonia (Mannheimia haemolytica, Pasteurella
multocida), liver abscess (Fusobacterium necrophorum), lymphadenitis
(Corynebacterium pseudotuberculosis, Streptococcus spp.), enteritis
(Escherichia coli, Salmonella spp.) and wound infections
(Staphylococcus aureus, Pseudomonas aeruginosa) (1). Infections that
involve a biofilm mode of growth are generally chronic and are often
difficult to treat (1,2,3,4).

Traditionally, microbiologists have evaluated the efficacy of an
antibiotic by measuring the minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) (5,6). In virtually all
diagnostic laboratories, these measurements are made on freely
floating, planktonic, laboratory phenotypes. These assays measure only
the concentration of chemotherapeutic agent required to inhibit growth
or kill planktonic bacteria (5,6). For some antibiotics, the
concentration required to kill sessile bacteria may be greater than a
thousand times that required to kill planktonic bacteria of exactly
the same strain (4,7). Therefore, the use of typical laboratory
planktonic bacteria for selection of chemotherapeutics may be
inappropriate under some circumstances.

We have recently developed a technology to screen the effectiveness of
antibiotics or biocides at eliminating sessile bacteria in vitro (8).
The use of the Calgary Biofilm Device (CBD) permits rapid selection of
potentially effective antibiotics for killing sessile bacteria in vivo
and biocides for disinfecting contaminated inert surfaces (8). This
device determines the minimum biofilm eradication concentration
(MBEC), which is the concentration of an antimicrobial agent required
to kill a bacterial biofilm. Recent studies conducted in our
laboratory have demonstrated that selecting antibiotics that are
effective for eliminating bacterial biofilms may improve the success
rate in treating clinical and experimentally induced disease (9). The
objective of this project was to determine culture conditions where
veterinary pathogens would form biofilms on the CBD. A second
objective was to evaluate the ability of antibiotics commonly used in
veterinary medicine to eliminate a diverse selection of bacterial
biofilms. Ultimately, the study was conducted to provide the basis of
future extensive screening of the susceptibility of antibiotics to
veterinary bacterial pathogens.
Top
Abstract
Introduction

Results
Discussion
References

Materials and methods

Organisms
Bacterial isolates were obtained from clinical cases of infections of
cattle, sheep, pigs, chickens and turkeys. Isolates were obtained from
the Animal Health Unit of the University of Calgary in Calgary,
Alberta and from Alberta Agriculture, Food and Rural Development in
Edmonton, Alberta. These isolates included: Corynebacterium
pseudotuberculosis, Corynebacterium renale, Mannheimia haemolytica,
Pasteurella multocida, Arcanobacterium (Actinomyces), Haemophilus
somnus, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus
hyicus, Streptococcus agalactiae, Streptococcus dysgalactiae,
Streptococcus suis, Escherichia coli and Salmonella spp.

Selection of antibiotics
The antibiotics evaluated were those commonly approved for the
treatment of bacterial infections in animals. The antibacterial agents
evaluated included: ampicillin, cloxacillin, erythromycin, gentamicin,
oxytetracycline, penicillin G, streptomycin, tetracycline (Sigma
Chemical Company, St. Louis, Missouri, USA), ceftiofur (The Upjohn
Corporation, Kalamazoo, Michigan, USA), enrofloxacin (Bayer Animal
Health, Kansas City, Kansas, USA), tilmicosin (Eli Lilly, Greenfield,
Indiana, USA), and trimethoprim-sulfadoxine (Hoechst Animal Health,
Regina, Saskatchewan).

Biofilm formation on the Calgary Biofilm Device
Biofilm formation and measurement of antimicrobial sensitivity of
bacterial biofilms were performed on the CBD (MBEC Biofilm
Technologies, Calgary, Alberta) according to previously described
methods (8). The device features a microtiter plate lid with 96 pegs
or projections distributed on the lid. Each peg provided the surface
for bacteria to adhere, colonize and form a uniform biofilm (8). The
pegs fit precisely into the wells of a standard 96-well microtiter
plate. The lid was used in conjunction with special troughs for
growing of bacteria, washing, and incubating. One of tryptic soy broth
(BDH), tryptic soy broth with bovine serum (Sigma Chemical Company) or
HS broth (Difco Laboratories, Detroit, Michigan, USA) for H. somnus
was placed in the trough. The trough was inoculated with approximately
108 test bacteria (based upon McFarlane standards) obtained from
colonies selected from tryptic soy agar (TSA, BDH) or brain heart
infusion (BHI, BDH) agar plates. The pegged lid was placed over the
troughs and the unit incubated on a rocker [Red Rocker; Hoefer
Instruments, San Francisco, California, USA; 10 rpm (2.5 × g)] at 37°C
and 95% relative humidity. The pegs were colonized for 4 to 24 h
(depending on the specific bacterial growth rate). Selection of
culture conditions for colonization of the pegs was determined in
preliminary studies and the assessment of biofilm was determined by
breaking several pegs from various points on the lid. The removed pegs
were placed in microfuge tubes containing 200 μL of TSB, sonicated
(Aquasonic, model 250; VWR Scientific, Buffalo Grove, Illinois, USA)
for 5 min and plate counts of viable bacterial cells were performed on
TSA or BHI agar containing 10% sheep blood. Additional pegs were fixed
with 2.5% gluteraldehyde in phosphate-buffered saline (PBS), air-dried
overnight, and prepared for scanning electron microscopy (SEM)
(Hitachi model 450; Hitachi, Tokyo, Japan), as described previously
(10).

Minimum biofilm eradication concentration assay
Assays were performed when pegs contained approximately 104 to 106
bacteria growing as a biofilm following conditions developed from the
procedure described above. By using SEM, we have established that
biofilms are produced at this level of peg colonization and we are not
studying adherent bacterial cells. These biofilms can then be used for
assessment of antimicrobial activities. Non-adherent bacteria on the
pegs were washed from the pegs in a 96-well microtiter plate
containing sterile PBSS. Each test antibiotic was placed in one lane
of the microtiter plate at 2-fold dilutions of antibiotic (from 1024
μg/mL to 2 μg/mL). Seven antibiotics were evaluated on each plate and
one lane served as a negative control (no antibiotic). All samples
were run in duplicate. Pegs with the bacterial biofilm were secured
over the test microtiter plate and the plate was incubated for 24 h at
37°C with antibiotic. The pegged lid was then removed, rinsed in PBS,
then placed over a second 96-well microtiter plate containing fresh,
sterile broth medium. The remaining biofilm was removed from the pegs
by ultrasonic disruption for 5 min. This plate was incubated for 24 h
at 37°C and the presence of viable bacteria determined by plate counts
or turbidity determined at 650 nm in a 96-well plate reader (Molecular
Devices; Fisher Scientific, Nepean, Ontario). Growth of bacteria in a
particular well indicates regrowth of planktonic bacteria from
surviving biofilm. Therefore, the MBEC value represents the lowest
dilution at which bacteria fail to regrow.

Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC), which represents the
concentration of antibiotic required to inhibit growth of a planktonic
bacterial population, was determined using the CBD. The MIC was
determined from the bacteria that were shed from the pegs of the CBD
when it was placed in the differing concentrations of antibiotics (8).
The MIC values obtained using the CBD are similar to those obtained
using the National Committee for Clinical Laboratory Standards (NCCLS)
procedure (8).
Top
Abstract
Introduction
Materials and methods

Discussion
References

Results

Bacterial biofilms were readily formed by most pathogens on the CBD
(Table I). The biofilms consisted of microcolonies encased in
extracellular polysaccharide material (1,4). Typical biofilms are
illustrated in Figures 1a, 1b, and 1c. Although bacteria grew readily
as planktonic organisms in liquid culture media, some bacteria would
not form biofilms under standard cultural conditions. Special
conditions were required for these organisms to grow as biofilms
(Table I). In order to form biofilms, C. renale, C.
pseudotuberculosis, M. haemolytica, P. multocida, and A. pyogenes
required the addition of fetal bovine serum and incubation under 10%
CO2. The duration of heavy biofilm formation on the pegs [> 104 colony-
forming units (cfu) per peg] varied from 4 h for P. aeruginosa to 24 h
for A. pyogenes, M. haemolytica, C. renale, and C. pseudotuberculosis.
Bacteria that required these more specialized culture conditions to
form biofilms also required longer culture time.
Table thumbnail
Table I.
figure 4FF1
Figure 1. Representative examples of biofilm formation of veterinary
pathogens. Staphylococcus aureus (mastitis isolate), Pasteurella
multocida (poultry isolate), and Corynebacterium renale (bovine
isolate) are demonstrated colonizing the peg of the CBD (more ...)

The concentrations of antibiotic required to inhibit planktonic
bacteria (MIC) and those required to kill biofilm bacteria (MBEC) are
summarized in Tables II and III. Most antibiotics were effective in
inhibiting planktonic bacterial growth at low concentrations and the
bacteria would be considered sensitive based upon NCCLS breakpoints
(11). Only a limited number of antibiotics were effective in killing
biofilm bacteria at relatively low concentrations. In some cases,
biofilm bacteria, such as A. pyogenes and S. aureus, appeared to lack
sensitivity to all of the antibiotics evaluated.
Table thumbnail
Table II.
Table thumbnail
Table III.

Gram-positive organisms growing as biofilms proved to be particularly
resistant to most antimicrobial agents. Most planktonically growing
organisms were sensitive to virtually all of the antibiotics tested
(Table II). Biofilms composed of A. pyogenes, S. aureus, S. hyicus, S.
agalactiae, C. renale, and C. pseudotuberculosis were highly resistant
to antimicrobial agents evaluated but sensitive to the tested agents
as planktonic bacteria. Both biofilm and planktonic forms of S.
dysgalactiae and S. suis were sensitive to the β-lactam drugs
(penicillin, ceftiofur, cloxacillin, ampicillin) and oxytetracycline.
Table thumbnail
Table II.

There was considerable variation in the results obtained for E. coli
isolates grown as sessile (biofilm) and planktonic populations (Table
III). Planktonic E. coli were sensitive to enrofloxacin, gentamicin,
oxytetracycline, and trimethoprim/sulfadoxine. Enrofloxacin and
gentamicin were the most effective antibiotics for E. coli growing as
biofilms. Salmonella growing as planktonic populations were sensitive
to enrofloxacin, gentamicin, ampicillin, oxytetracycline, and
trimethoprim/sulfadoxine. When Salmonella spp. were grown as biofilms,
they were only sensitive to enrofloxacin and ampicillin (Salmonella
Bredeny only). Planktonic and biofilm populations of P. multocida and
M. haemolytica had similar antibiotic sensitivity profiles with the
exception of trimethoprim/sulfadoxine. Planktonic P. aeruginosa were
sensitive to enrofloxacin, erythromycin, and oxytetracycline. The
sessile forms of this organism were sensitive only to enrofloxacin.
Both planktonic and sessile H. somnus were sensitive to enrofloxacin,
gentamicin, erythromycin, tilmicosin, ampicillin, oxytetracycline, and
trimethoprim/sulfadoxine.
Table thumbnail
Table III.
Top
Abstract
Introduction
Materials and methods
Results

References

Discussion

The MIC has been used as a gold standard for determination of
antimicrobial sensitivities for animal and human pathogenic bacteria
(4,5). It is recognized that an antibiotic that is ineffective in
preventing growth of a particular organism using the MIC assay will
also be clinically ineffective (12). However, an organism that is
sensitive in vitro may not be effective in vivo (12,13,14,15,16). For
many veterinary bacterial diseases the MIC value for a particular
antibiotic is not predictive of clinical efficacy. Nevertheless, up to
this time, the MIC assay remains the best way to select potentially
effective antimicrobial agents. The CBD and the MBEC assay were
developed for rapid and reproducible antimicrobial susceptibility
testing for bacterial biofilms in the anticipation that the MBEC would
be more reliable for selection of clinically effective antibiotics.

In human medicine it has been estimated that 65% of nosocomial
infections are biofilm associated, costing the health care system
billions of dollars (1,3,15). These biofilm infections are 10 to 1000
times more resistant to the effects of antimicrobial agents (1,3,7).
Indeed, many veterinary bacterial pathogens exist predominantly as
adherent (also called biofilm or sessile) organisms within tissue and
on inert surfaces and it is well recognized that such infections are
extremely difficult to successfully treat (14,15,17,18). The mechanism
for enhanced antimicrobial resistance is believed to involve
alterations in gene expression leading to a phenotype difference
between the planktonic and sessile forms. The sessile forms are more
resistant as they produce exopolysaccharde, have different growth
characteristics and take up nutrients and drugs differently from their
planktonic counterpart (3,16). The CBD was developed to address the
issues of enhanced antimicrobial resistance within biofilms.
Determination of MBEC might, therefore, permit selection of a
particular antibiotic that would more closely reflect the prognosis of
antimicrobial therapy for a particular bacterial infection.

Staphylococcus biofilms have been extensively studied in human
medicine and this pathogen is considered significant in both device
associated infections and tissue infections such as pneumonia and
osteomyelitis (1,19). The prevalence of bovine staphylococcal mastitis
ranges from 7% to 40% of all dairy cattle and this infection is
associated with bacterial biofilms (14,17,18,19). It is also
recognized that antibiotic therapy may temporarily eliminate clinical
signs of mastitis but the prognosis of a complete cure is poor (14).
Although, in this study, the MIC assay clearly indicated that many
antibiotics should be effective in the treatment of bovine mastitis,
the MBEC values data demonstrated that the S. aureus isolate is
resistant to antibiotics tested, correlating with clinical
observations. The S. hyicus biofilm as measured by MBEC was sensitive
to many of the antibiotics tested; indeed, S. hyicus usually responds
well to antibiotic therapy (20).

There is considerable variability in therapeutic responses to
streptococcal infections (20). S. suis and S. dysgalactiae infections
frequently respond readily to most chemotherapeutic agents (20).
Although these organisms formed biofilms, the MIC and MBEC values were
similar, suggesting that most of the antibiotics evaluated would be
effective as chemotherapeutic agents. The S. agalactiae isolate
studied, recovered from an animal with chronic mastitis, was sensitive
to most antibiotics according to the MIC, but as a biofilm, it was
resistant. S. agalactiae mastitis is highly infectious and usually
responds to treatment. As this isolate was recovered from an animal
with chronic unresponsive mastitis, it may be genotypically and
phenotypically altered to be resistant as a biofilm (3,16).

Although C. renale, C. pseudotuberculosis and A. pyogenes grew readily
as planktonic bacteria in enriched broth, they required specific
culture conditions, such as addition of serum to the media and
culturing under increased carbon dioxide concentration, to induce
formation of biofilms. This suggests that for some microorganisms
simulation of the growth conditions that exist in the host may be
required. These organisms were sensitive to all antimicrobial agents
tested (except streptomycin) according to the MICs, but they were
highly resistant according to the MBEC values. The MBEC values appear
to be more predictive, as infections caused by C. renale, C.
pseudotuberculosis, and A. pyogenes require prolonged antimicrobial
therapy and are frequently unresponsive to treatment (20). The ability
of biofilm bacteria to avoid phagocytosis by macrophages and
neutophils may also account for the abscessation observed within these
infections (1). The accumulation of pus and the associated
encapsulation of the infection site also inhibits the antimicrobial
penetration and pathogen destruction.

Most gram-negative livestock pathogens readily form bacterial biofilms
and these biofilms have been previously described in livestock
infections such as neonatal colibacillosis (21) and pneumonic
pasteurellosis (18). The veterinary E. coli isolates tested readily
formed biofilms on the CBD and with the exception of enrofloxacin,
gentamicin, and ampicillin, these biofilms were resistant to the
antibiotics tested. This suggests that once E. coli biofilms have been
established, they may be difficult to treat with some antibiotics.
This has been observed in some clinical cases in cattle, swine and
poultry (20,22). There was considerable variability among the MICs of
the Salmonella spp. isolates in this study: similar variability
between the MIC and the MBEC values was observed. This observation may
reflect the complexity in prediction of chemotherapeutic agents for
treatment of different Salmonella isolates. Bovine, porcine, and avian
Pasteurella spp., as well as the H. somnus and M. haemolytica isolate
tested, formed biofilms, but in most cases there was no difference
between the MIC and the MBEC values. Indeed, animals with
pasteurellosis or hemophilosis respond well to most antimicrobial
agents provided that a secondary pathogen (A. pyogenes, S. aureus) is
not involved (20).

Pseudomonas aeruginosa has been recognized in human medicine to form
antibiotic resistant biofilms on implanted devices and within tissues
(3). Pseudomonas aeruginosa infections in animals are similarly
difficult to treat (23). The planktonic Pseudomonas isolate was
resistant to most antibiotic agents, but biofilm cells were more
resistant and only enrofloxacin demonstrated reasonable clinical
activity. Fluorinated quinolones have been shown to be effective in
treatment of most Pseudomonas infections (1,3).

Planktonic bacterial sensitivity, pharmacokinetics, drug penetration,
local activity, and drug inactivation all influence the clinical
efficacy of an antibacterial agent, but to date, the efficacy of
veterinary antibiotics in elimination of bacterial biofilms has not
been evaluated. The CBD and the MBEC assay provide a new technology
that can be used to select antibiotics that are effective in killing
biofilm bacteria. This new technology can also be used in the
pharmaceutical industry for developing new antimicrobial agents with
efficacy against bacteria growing as biofilms (8). Recently, we have
used the MBEC assay to predict clinical failure and clinical success
of certain antibiotics used to treat peritonitis due to device-
associated infections in humans (24). It may be possible to apply this
technology in veterinary bacterial infections that are difficult to
treat. This study was conducted to demonstrate the diversity of
organisms that could form biofilms that were resistant to common
veterinary antibiotics. Further studies are required to document
variations within a specific species or from a defined bacterial
disease.

Footnotes
Acknowledgments

This research was supported by the Alberta Agriculture Research
Institute and the Natural Sciences and Engineering Research Council.
The authors acknowledge the technical support of Carol Ann Stremick
and Liz Middlemiss.

Address correspondence and reprint requests to Dr. Merle E. Olson,
telephone: 403-220-6836, fax: 403-270-0954, e-mail: molson@ucalgary.ca

Received July 27, 2001. Accepted November 29, 2001.
Top
Abstract
Introduction
Materials and methods
Results
Discussion

References
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The take home here is that it does not have to be MSRA to be resistant
to antibotics.
Ordinary Staph A. is resistant to all the antibiotics tested here once
it settles down to biofilm form, and amoxicillian while a great drug
for sepsis, is use against sinusitis pathogens line Staph A and
Pseudomondas.

In another artical Linezolid was tested in the Calgery Biofilm Devise
and found effective.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=226988