This article is discussed in the editorialavailable at: http://dx.doi.org/10.1007/s00134-008-1101-0.
L. Berra (�)Massachusetts General Hospital, HarvardMedical School, Department of Anesthesiaand Critical Care,55 Fruit Street, Boston WHT-4-436, MA,USAe-mail: [email protected]
F. Curto · G. Li Bassi · T. KolobowNational Institutes of Health, Pulmonaryand Critical Care Medicine Branch, NHLBI,9000 Rockville Pike, Bethesda 20892, MD,USA
P. LaquerriereUniversity of Reims Champagne Ardenne,INSERM-ERM 0203, Laboratoire deMicroscopie Electronique, UFR Sciences,21 rue Clement Ader, BP 138, 51685Reims, Cedex 2, France
B. PittsMontana State University Bozeman, Centerfor Biofilm Engineering and Department ofChemical Engineering,Bozeman 59717-3980, MT, USA
A. BaccarelliHarvard School of Public Health,Department of Environmental Health,Boston 02215, MA, USA
Abstract Objective: Antibiotic-resistant bacterial biofilm mayquickly form on endotracheal tubes(ETTs) and can enter the lungs,potentially causing pneumonia. Inan attempt to prevent bacterial colo-nization, we developed and tested inan in-vitro study and animal studyseveral antibacterial-coated ETTs(silver sulfadiazine with and with-out carbon in polyurethane, silversulfadiazine and chlorhexidine withand without carbon in polyurethane,silver–platinum with and withoutcarbon in polyurethane, chlorhexidinein polyurethane, and rose bengal forUV light). Design, setting, animals,interventions: After preliminary stud-ies, silver sulfadiazine in polyurethane(SSD-ETT) was selected among thecoatings to be challenged every 24 hwith 104–106 Pseudomonas aerug-inosa/ml and evaluated at 6 h, 24 h,and 72 h with standard microbio-logical studies, scanning electronmicroscopy, and confocal scanning
microscopy. Subsequently, eightsheep were randomized to receiveeither a SSD-ETT or a standard ETT(St-ETT). After 24 h of mechanicalventilation, standard microbiologicalstudies were performed together withscanning electron microscopy andconfocal microscopy. Measurementsand results: In the in-vitro studySSD-ETT remained bacteria-free forup to 72 h, whereas St-ETT showedheavy P. aeruginosa growth andbiofilm formation (p < 0.01). Insheep, the SSD-ETT group showed nobacterial growth in the ETT, ventilatortubing, and lower respiratory tract,while heavy colonization was foundin the St-ETT (p < 0.01), ventilatortubing (p = 0.03), and lower respira-tory tract (p < 0.01). Conclusion:This study describes several effectiveand durable antibacterial coatings forETTs. Particularly, SSD-ETT showedprevention against P. aeruginosabiofilm formation in a 72-h in-vitrostudy and lower respiratory tractcolonization in sheep mechanicallyventilated for 24 h.
Nosocomial pneumonia is one of the leading causes ofmorbidity and mortality in hospitalized patients [1, 2].While a number of factors contribute to the increased riskof nosocomial pneumonia in patients in the intensive careunit, mechanical ventilation using an endotracheal tube(ETT) represents one of the greatest risks [3–5].
Recent studies showed that antimicrobial-coated ETTmay be used to lower lung colonization, claiming tolower the incidence of ventilator-associated pneumonia(VAP) [6–14]. In the design of antimicrobial-impregnatedbiomaterials, both the selection of the type and the rate ofrelease of the antimicrobial agent from the medical deviceare important. Ideally, the selected antimicrobial agentshould possess a lasting broad-spectrum antimicrobial ac-tivity and a low degree of bacterial resistance. The wide-spread occurrence of antibiotic resistance is alarming [15,16]; hence, an interest has emerged in the use of medicaldevices coated with nonantibiotic antimicrobial agents.
We explored several biomaterials and fabricated tendifferent coatings for ETT. After testing the tubes foreffectiveness and feasibility, we selected the coating withsilver sulfadiazine (SSD-ETT). In an in-vitro experiment,we challenged SSD-ETT and non-coated ETT with Pseu-domonas aeruginosa to: (a) compare the bacterial growthrate; (b) determine the ability of silver sulfadiazine toprevent bacterial attachment to ETT; and (c) determine theability of silver sulfadiazine to kill pathogenic bacteria.Secondly, we tested SSD-ETT in sheep to: (a) assess thebactericidal effects of the SSD coating in the ETT andthroughout the ventilator circuit; (b) measure the reductionin bacterial colonization of the lungs; and (c) investigatelocal and systemic side effects.
Materials and methods
ETT coatings: materials and fabrication
In a laboratory at the US National Institutes of Health,Bethesda, Maryland, we fabricated ten bacteriostatic andbactericidal ETT coatings: (1) chlorhexidine in poly-urethane, (2) silver sulfadiazine in polyurethane, (3) silversulfadiazine and chlorhexidine in polyurethane, (4) silversulfadiazine and carbon in polyurethane, (5) silver sulfadi-azine chlorhexidine and carbon in polyurethane, (6) silverin polyurethane, (7) silver and carbon in polyurethane, (8)silver–platinum in polyurethane, (9) silver–platinum andcarbon in polyurethane, and (10) rose bengal for UV light(see Fig. 1).
Silver sulfadiazine and chlorhexidine
The silver salt of sulfadiazine with or without chlorhex-idine was developed recently. It is widely used to coat
Fig. 1 Coated endotracheal tubes. From left to right: 1, standardnon-coated ETT; 2, silver sulfadiazine chlorhexidine and carbonin polyurethane; 3, silver sulfadiazine and carbon in polyurethane;4, silver sulfadiazine and chlorhexidine in polyurethane; 5, silversulfadiazine in polyurethane; 6, chlorhexidine in polyurethane;7, standard non-coated ETT; 8, rose Bengal for UV light;9, silver and carbon in polyurethane; 10, silver in polyurethane;11, silver–platinum in polyurethane; 12, silver–platinum and carbonin polyurethane
medical devices to prevent catheter-related infections andis currently the treatment of choice for burn wounds, asit has activity against gram-negative and gram-positivebacteria, fungi, protozoa, and certain viruses. However,the mechanism of silver sulfadiazine and chlorhexi-dine’s antibacterial action has not been fully elucidated.After exposure, structural changes occur in the bacterialcell membrane, such as distortion and enlargement ofthe cell and weakening of the membrane. Silver sul-fadiazine molecules dissociate, and the silver moietyenters the cell wall, attaches to the DNA, and preventsbacterial cell proliferation. Chlorhexidine alters the cellmembrane sufficiently to permit the efflux of nitrogenbases, nucleotides, and nucleosides and facilitate entry ofsulfadiazine molecules [17–19].
We coated the lumen of ETT with chlorhexidine inpolyurethane; silver sulfadiazine in polyurethane; silversulfadiazine and chlorhexidine in polyurethane; silversulfadiazine and carbon in polyurethane; and silver sulfa-diazine, chlorhexidine, and carbon in polyurethane (Fig. 1,coated ETT nos. 2–6)
Oligodynamic iontophoresis
One of our early research paths was directed towards elec-trically injecting metal ions (silver) into solution. This hasbeen shown to reduce bacterial colonization 15- to 100-fold in both bench-top and animal experiments. Bacterici-dal iontophoretic polymers can be designed to release sil-ver ions when moistened with body fluids in the presenceof silver and platinum powder. When the composite ma-terial is placed in contact with or immersed in an electri-cally conductive medium, such as saline, blood, or urine, ormucus, the metal powder becomes an array of small elec-trodes. Specifically, each metal granule embedded in thebase material becomes either an anode or a cathode. Mi-crobial growth is impaired through release of silver ions,
1022
with the generation of electric current from 1 to 400 µA.This range of current does not cause localized cell necrosisand is below the sensory or pain threshold. The amounts ofcarbon, silver, and platinum powder, their ratios, their par-ticle size and the permeability of the polymer compositionall affect the rate of silver ion release [20].
We coated the lumen of ETT with silver in poly-urethane; silver and carbon in polyurethane; silver–plat-inum in polyurethane; and silver–platinum and carbon inpolyurethane (Fig. 1, coated ETT nos. 9–12).
Photodynamic therapy
Rose bengal is a perhalogenated fluorescein derivative thatis among the most efficient known producers of singletoxygen. Activation with UV light causes singlet oxygenproduction and photosensitization. Rose bengal has beenwidely used in photodynamic therapy of tumors, to inacti-vate viruses, gram-positive bacteria, and protozoa, and toproduce photohemolysis, and to induce occlusion of bloodvessels in a procedure called photothrombosis [21]. Wecoated the lumen of ETT with rose bengal (Fig. 1, coatedETT no. 8). During mechanical ventilation, a probe wasconnected to an UV-visible light source and introduced in-side the ETT.
Coating selection
After having selected the antimicrobial agents and concen-trations, we tested the efficacy and safety of the variouslycoated ETT in repeated in-vitro and animal studies overa period of approximately 2 years. All of them showed bac-teriostatic and bactericidal effects [11, 22–24].
However, we selected the silver sulfadiazine inpolyurethane coating for further investigation, because:
a) SSD is widely and safely used in the clinical set-ting (i. e., cream preparation, IV catheters, urinarycatheters, prostheses).
b) Unlike UV light, SSD-ETT do not require extra care.c) The SSD coating is very smooth and resistant to torque.d) At extubation after prolonged mechanical ventilation in
sheep (up to 7 days of intubation) the SSD coating ap-peared to maintain its characteristics on visual inspec-tion and on microscopy (Fig. 2a, b).
e) One year after the coating process, the SSD coating re-tained antibacterial properties in repeated studies. Elec-tron microscopy and confocal laser microscopy showedno morphological differences from new, unused SSD-ETT.
The coating procedure
We prepared a dispersion of 53 g of silver sulfadiazine,and 22.5 g of polyurethane (BioSpan) in 210 ml of N, N-
Fig. 2 Silver sulfadiazine in polyurethane coated ETT. a Lumen ofa SSD-ETT after 7 days of intubation and mechanical ventilation insheep. The coating looks like the new, never-used coated SSD-ETT.b Scanning electron microscopy: cross-section of the same tube
dimethylacetamide. We inserted a standard 8-mm trachealtube (Lo-Contour TM, Mallinckrodt, St. Louis, USA) intoa hollow transparent acrylic tube, to keep the ETT straight.With the plastic tube positioned vertically, we immersedthe ETT tip into the dispersion, rapidly aspirated the dis-persion up to the level of the connector piece, and thenlet the ETT drain for 2–4 s. Then we placed the transpar-ent plastic tube with the ETT horizontally into a rotatingdevice, through which a stream of air was gently passedto dry the dispersion. After 12 h, the coated ETT was re-moved and sterilized with ethylene oxide gas. Pictures ofthe lumen of the SSD-ETT are shown in Fig. 2.
In-vitro study
In a set of experiments (six replicates), 104–106 P. aerug-inosa/ml in biofilm medium were placed in the lumen ofSSD-ETT and standard non-coated ETT (St-ETT). StrainPaO1 containing the GFP-plasmid (pMRP9-1 carbenicillinresistant) [25] was used to: (a) determine the ability ofthe silver sulfadiazine coating to prevent bacterial attach-ment and ETT-biofilm formation and (b) compare bacterialgrowth rates in biofilm medium.
For the growth medium, carbenicillin powder was di-luted to 150 µg/ml using 1% Trypticase Soy Broth solvent.This was used to dilute P. aeruginosa PA01 to 105–107
cells/ml. St-ETT and SSD-ETT were clamped 1 cm prox-imal from the inflatable cuff and partially filled with 8 mlof freshly prepared biofilm medium. Tubes were clamped2 cm from the ETT connector piece and incubated at 37 °Cwith mild shaking.
The bacterial challenge was stopped at 6, 24, and 72 h.Bacterial count of ETT broth was calculated using standardmicrobiology methods for bacterial quantification counts.Scanning electron microscopy of the ETT lumen was per-formed to assess the biofilm and the thickness of secre-tions. Using aseptic technique, a 1-cm-long cross sectionof ETT was excised and placed in a sterile vial filled with2.5% glutaraldehyde, and stored at 4 °C for scanning elec-tron microscopy (SEM) and confocal laser scanning mi-croscopy (CLSM) [11].
1023
Animal laboratory study
The study was conducted and approved by the National In-stitutes of Health.
The SST-ETT animal study was performed in sheep toassess both (a) prevention of bacterial colonization of therespiratory tubing and of the lower respiratory tract and (b)local and systemic side effects in an animal model.
This 24-h study involved eight young female Dorsetsheep. Sheep were randomized to receive either a standardETT (n = 4) or an ETT internally coated with silver sul-fadiazine in polyurethane (n = 4), and were mechanicallyventilated for 24 h. The number of sheep for each groupwas calculated based on the results of our previous studyon coated ETT in animals mechanically ventilated for 24 h.The protocol, preparation, and monitoring of the animalstudy were described previously [11].
At autopsy, the thorax was opened using strict asep-tic techniques, and the lungs were exposed, excised, andweighed. Tissue samples were collected for quantitativeculture from each lobe and the lobar bronchi. A total of 12tissue samples weighing approximately 50 mg each weretaken: five samples from the five lobes of the lungs, fivesamples from the five corresponding lobar bronchi, onesample from the trachea 2 cm above the carina, and onesample from the middle part of the ETT (Fig. 3a). The oralcavity was sampled at the beginning of the study. The tra-chea and the larynx were opened through a longitudinalanterior-midline–incision up to the carina for visual in-spection of the mucosa and of the ETT. The trachea wasexcised and sent for microscopic study. All tissue/mucusand fluids were sent for quantitative and qualitative aer-obic cultures using standard bacteriologic techniques. Allindwelling devices were cultured.
Table 1 In-vitro study: scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and microbiology findings inETT challenged with P. aeruginosa after 6 h, 24 h, and 72 h
6 h 24 h 72 hSSD-ETT St-ETT SSD-ETT St-ETT SSD-ETT St-ETT
SEMAbsence of bacteria × × ×Adhesion of bacteria ×Formation of microcolonies ×Confluent colonies ×Thickness (µm) 0 0–2 0 10–30 0 15–50
CLSMAbsence of bacteria × × ×Adhesion of bacteria ×Formation of microcolonies ×Confluent colonies ×Thickness (µm) 0 0–5 0 20–50 0 30–70
* p-values calculated using Fisher’s Exact test: SSD-ETT vs. St-ETT
The internal lumen of the ETT was sampled 10–12 cmfrom the ETT connector piece every 8 h (four samples) forbacterial growth using a cotton culture swab.
Just before the end of the study, we sampled the airfilter between the ventilator and the humidifier, the waterfrom the humidifier, the inspiratory and expiratory linesof the mechanical ventilator approximately 10–12 cm fromthe ETT connector piece, and the expiratory line conden-sate water trap (Fig. 3b).
Statistical analysis
We used the Wilcoxon (Mann–Whitney) rank sum test forgroup comparisons of continuous variables. Fisher’s exact
Fig. 3 Animal study: sample sites. a Silicon rubber cast of sheeplungs. White circles indicate sites from which samples were takenupon autopsy for microbiological studies: trachea, five bronchi, andfive lobes of the lungs (from Panigada et al., Crit Care Med 2003;31:729–737). b Ventilator circuit of the sheep study, and samplesites: a, Swabs from the air filter, humidifier, inspiratory lines, ETT,expiratory lines, and water trap; b, ETT biofilm scraped for lightmicroscopy studies; c, Secretions from inside the ETT for bacterialculture (CFU/g); d, Two rings of the ETT were cut, one for confo-cal scanning laser microscopy studies and one for scanning electronmicroscopy [11]
1024
test was used for the analysis of categorical variables. A p-value < 0.05 was considered statistically significant. Alltests were two-sided. We performed all analyses with theStata statistical package (Stata, College Station, TX; re-lease 8.0).
Results
In-vitro study
SEM revealed bacterial adhesion on the St-ETT poly-vinylchloride at 6 h (Fig. 4a), formation of microcoloniesat 24 h, and uniform protein-like deposits at 72 h (Fig. 4b)(thickness of secretions on the lumen of St-ETT rangedfrom 0 µm to 70 µm; Table 1). No bacteria or secretionswere detected at any time on SSD-ETT (Fig. 4c, d;Table 1). The culture broth was always heavy colonized inthe St-ETT (bacterial growth during the 72 h study periodranged from 3.1 × 104 to 3.2 × 109 cfu/g), while in theSDD-ETT bacteria were present only in two samples after24 h (bacterial growth during the 72 h study period rangedfrom 0 to 3.5 × 107 cfu/g) (Table 1).
Animal study
All study animals were healthy upon enrollment, based onclinical findings, laboratory data, and chest X-ray during
Fig. 4 In-vitro study: SEMmicrographs. ETT samples wereimaged with scanning electronmicroscopy. a After 6 h ofchallenge with P. aeruginosa,bacteria were seen to adhere tothe polyvinylchloride lumen ofthe St-ETT. b After 72 h, a thickbiofilm covered the entire tubesurface. A chain of bacteria canbe seen emerging from the thickbiofilm. c, d No bacteria wereseen to adhere at any time to thecoated surface of SSD-ETT(c after 6 h; d after 72 h). Thesilver sulfadiazine coating has agranular appearance
24 h of mechanical ventilation. Intubation was successfulat the first attempt in all sheep. The PaO2/FiO2 ratiowas greater than 400 at all times in all sheep. No fever,purulent secretions in ETT, abnormal leukocyte counts, orchanges on chest radiographs were observed. At autopsy,no gross abnormalities were identified in the trachealmucosa.
In the St-ETT group, the lower respiratory tractand ventilator tubing were extensively colonized (lowerrespiratory tract colonization ranged from 5.0 × 105 to5.5 × 108 cfu/g). No bacterial colonization was detectedthroughout the lower respiratory circuit, and ventila-tor tubing (lower respiratory tract colonization ranged0–0 cfu/g, p < 0.01 vs. St-ETT) in sheep intubated withthe SSD-ETT (Table 2).
Microscopic studies showed a thick and dense secre-tion layer covering the lumen of the St-ETT (range50–750 µm, bacterial colonization 5.0 × 106–3.5× 108 cfu/g) (Table 2, Fig. 5a), and at higher magnifi-cation accumulations of bacteria, white blood cells, andred blood cells could be easily identified (Fig. 5b). OnSDD-ETT, the mucus layer was much thinner, rangingfrom 0 to 450 µm, and no bacteria were observed (bacte-rial colonization 0–0 cfu/g, p < 0.01 vs. St-ETT) (Table 2;Fig. 5c, d). The most common aerobic bacteria found inthe oral secretions were α-hemolyticStreptococcus spp.,Moraxella spp., Pasteurella spp., and Staphylococcusaureus; in addition to those bacteria, from the lowerrespiratory system of the Et-ETT group we cultured Kleb-
1025
Table 2 Animal study: microscopy and bacteriology findings in mechanically ventilated sheep intubated with SSD-ETT and St-ETT
SSD-ETT St-ETT p-value
SEM-CSLMThickness of secretion layer, min min–max, µm 25, 0–100 100, 50–200Thickness of secretion layer, max min–max, µm 250, 150–450 685, 650–750Well-defined biofilm architecture (n = 4) 0 0 1.00 *presence of bacteria (n = 4) 0 3 0.14 *Presence of red cells (n = 4) 1 2 1.00 *Presence of white cells (n = 4) 1 3 0.49 *
* p-values were calculated using Fisher’s exact test; ** p-values were calculated using Wilcoxon (Mann–Whitney) rank sum test; The mostcommon aerobic bacteria detected included: α-hemolytic Streptococcus spp. (not Streptococcus pneumoniae), Klebsiella pneumoniae,Moraxella spp, Pasteurella haemolytica, Pasteurella multocida, Pasteurella spp, P. aeruginosa, Staphylococcus aureus
Fig. 5 Animal study: SEMmicrographs. ETT samples wereimaged with scanning electronmicroscopy. a Note the thick(almost 700 µm) deposits on thelumen of St-ETT of sheep thatwere intubated and mechanicallyventilated for 24 h. b At highermagnification, red cells can beeasily recognized. c, d Nosecretions accumulated onSSD-ETT. The arrow (c) showsthe thickness of the coating(approximately 40 µm)
1026
Tabl
e3
Coa
ted
ando
trac
heal
tube
stud
ies
Pub
lica
tion
Mat
eria
lsfo
rS
tudy
-len
gth
Con
clus
ion
ofth
est
udy
the
ET
Tco
atin
g
In-v
itro
stud
ies
Har
tman
net
al.[
6]S
ilve
rba
sed
50h
Sil
ver-
coat
edE
TT
show
eda
sign
ifica
ntin
hibi
tion
ofgr
owth
ofP
seud
omon
asae
rugi
nosa
inth
eco
ntin
uous
lyco
ntam
inat
edan
dm
echa
nica
lly
vent
ilat
edor
opha
rynx
–lar
ynx–
lung
mod
el.
Sil
ver-
coat
edE
TT
thus
mig
htbe
help
fuli
nre
duci
ngV
AP.
Jone
set
al.[
7]H
exet
idin
e-im
preg
nate
dE
TT
8h
Bas
edon
the
impr
essi
vem
icro
bial
anti
-adh
eren
cepr
oper
ties
and
dura
bili
tyof
the
surf
acta
ntco
atin
gon
PV
Cfo
llow
ing
dip
coat
ings
,iti
spr
opos
edth
atth
ese
syst
ems
may
usef
ully
redu
ceth
ein
cide
nce
ofve
ntil
ator
-ass
ocia
ted
pneu
mon
iaw
hen
empl
oyed
aslu
min
alco
atin
gsof
the
ET
T.B
alaz
set
al.[
8]C
hem
ical
mod
ifica
tion
of72
hT
hech
emic
alm
odifi
cati
ons
usin
gN
aOH
and
AgN
O(3
)w
ettr
eatm
ents
com
plet
ely
inhi
bite
dE
TT-
PV
Cw
ith
sodi
umhy
drox
-ba
cter
iala
dhes
ion
of4
stra
ins
ofP.
aeru
gino
sato
both
nativ
ean
dox
ygen
-pre
-fun
ctio
nali
zed
ide
and
silv
erni
trat
eso
luti
ons
PV
C,a
ndef
fici
entl
ypr
even
ted
colo
niza
tion
over
72h.
Pach
eco-
Fow
ler
etal
.[14
]E
TT
sim
preg
nate
dw
ith
5da
ysA
ntis
epti
cE
TT
spr
even
ted
bact
eria
lcol
oniz
atio
nin
the
airw
aym
odel
and
also
reta
ined
chlo
rhex
idin
ean
dsi
gnifi
cant
amou
nts
ofth
ean
tise
ptic
.si
lver
carb
onat
eC
haib
anet
al.[
9]G
enti
anvi
olet
3w
eeks
Coa
ted
ET
Tim
preg
nate
dus
ing
anin
stan
tane
ous
dip
met
hod,
wer
esh
own
toha
vean
dch
lorh
exid
ine
broa
d-sp
ectr
umac
tivity
,pro
long
edan
timic
robi
aldu
rabi
lity
and
high
effi
cacy
inin
hibi
ting
adhe
renc
eof
orga
nism
sco
mm
only
caus
ing
noso
com
ial
pneu
mon
ia.F
urth
erm
ore,
thes
eco
ated
devi
ces
wer
esh
own
tobe
non-
cyto
toxi
c.
Ani
mal
stud
ies
Ols
onet
al.[
10]
Silv
erba
sed
96h
The
sere
sults
sugg
estt
hatt
hesi
lver
coat
ing
ofE
TT
sm
ayde
lay
the
onse
tof
and
decr
ease
the
seve
rity
oflu
ngco
loni
zati
onby
aero
bic
bact
eria
.B
erra
etal
.[11
]S
ilver
sulf
adia
zine
24h
Coa
ted
ET
Ts
indu
ced
ano
nsig
nifi
cant
redu
ctio
nof
the
trac
heal
colo
niza
tion,
elim
inat
edan
dch
lorh
exid
ine
(sev
enof
eigh
t)or
redu
ced
(one
ofei
ght)
bact
eria
lcol
oniz
atio
nof
the
ET
Tan
dve
ntil
ator
circ
uits
,and
prev
ente
dlu
ngba
cter
ialc
olon
izat
ion.
Clin
ical
tria
lR
ello
etal
.[13
]S
ilve
rba
sed
upto
Inth
ispr
ospe
ctiv
ely
plan
ned,
prel
imin
ary
anal
ysis
,the
ET
Tco
ated
was
feas
ible
and
wel
l32
days
tole
rate
d.L
arge
rst
udie
sar
ene
eded
tode
term
ine
whe
ther
dela
yed
colo
niza
tion
,red
uced
colo
-ni
zati
onra
te,a
ndde
crea
sed
bact
eria
lbur
den
wil
ldec
reas
eth
ein
cide
nce
ofV
AP.
1027
siella pneumoniae, Pasteurella haemolytica, Pasteurellamultocida, and P. aeruginosa.
Discussion
The incorporation of an antimicrobial agent within the con-stituent polymer of a medical device is an accepted methodto decrease the incidence of device-associated infection.This approach may reduce microbial adherence to the bio-material by virtue of an antimicrobial surface, as well as bythe release of drug into the surrounding medium in quanti-ties sufficient to achieve microbial killing. The inclusion ofantimicrobial agents to the component polymers of medi-cal devices has resulted in improved outcomes when usingdevices such as central venous catheters, urinary catheters,bone cements, cerebrospinal fluid shunts, and continuousperitoneal dialysis catheters [26–29].
Therefore, prevention of bacterial colonization of thepolyvinyl chloride (PVC) ETT has been suggested to lowercontamination of the lower respiratory system, and thusthe incidence of VAP. Many investigators have recently fo-cused their research on novel materials to coat the ETT toprevent bacterial colonization. Table 3 summarizes someof those studies.
In 1999, Hartmann et al. published the first study oncoated ETT [6]. The ETT was covered under vacuumwith 0.15- to 0.25-µm-thick silver films after pre-coatingwith different precious metals. They developed anoropharynx–larynx–lung model which was continuouslycontaminated with P. aeruginosa and which for the pur-pose of clinical simulation was mechanically ventilatedfor a period of 50 h. Coated ETTs showed significantreduction of bacterial counts in the oropharynx–larynxmodel throughout. In 2001, Jones et al. conducted anin-vitro study with hexetidine-impregnated PVC ETT [7].PVC emulsion was cured in the presence of hexetidine.The purpose of the study was to examine hexetidine-impregnated PVC ETT biomaterials with respect to theirtensile, surface, and drug release properties, and also theirresistance to the adherence of clinical isolates of Staphy-lococcus aureus and P. aeruginosa. ETT PVC containinghexetidine significantly lowered the number of adherentviable bacteria. In a fascinating study in 2004, Balazs et al.incorporated monovalent silver into the PVC [8]. The an-tiadhesive and antibacterial properties of this new materialwere tested on P. aeruginosa. The chemical modificationconsisted of a radiofrequency-oxygen glow discharge pre-functionalization, followed by a two-step wet treatment inNaOH and AgNO3 solutions. The creation of silver salton native PVC resulted in an ultrahydrophobic surface.The chemical modifications using NaOH and AgNO3wet treatments completely inhibited bacterial adhesionof four strains of P. aeruginosa and efficiently preventedcolonization for 72 h. In the same year, Pachego-Fowleret al. published a study employing chlorhexidine and
silver carbonate-coated ETT [14]. The antiseptic ETTwere compared with non-coated ETT to evaluate thepotential effectiveness of impregnating ETT with an-tiseptic to reduce colonization of methicillin-resistantS. aureus, P. aeruginosa, Acinetobacter baumannii, andEnterobacter aerogenes. In an in-vitro model the authorsshowed that antiseptic ETT significantly decreased orprevented bacterial colonization for 5 days in the trachealmodel and retained substantial amounts of the antisepticagents. There are only two animal studies exploring thebenefits of coated ETT. We showed that silver sulfadiazineand chlorhexidine in polyurethane decreases bacterialcolonization of the lower respiratory tract of sheep andventilator circuit after 24 h of mechanical ventilation [11].In a recent study, Olson et al. explored the benefits ofhydrogel silver-coated ETT (C.R. Bard) on 12 dogschallenged with P. aeruginosa [10]. They found that silvercoating of ETT delayed the appearance of bacteria on theinner surface of the ETT and decreased lung bacterialcolonization.
In our laboratory, we developed 10 different coatingsfor ETTs: (1) chlorhexidine in polyurethane, (2) silversulfadiazine in polyurethane, (3) silver sulfadiazine andchlorhexidine in polyurethane, (4) silver sulfadiazine andcarbon in polyurethane, (5) silver sulfadiazine chlorhexi-dine and carbon in polyurethane, (6) silver in polyurethane,(7) silver and carbon in polyurethane, (8) silver–plat-inum in polyurethane, (9) silver–platinum and carbonin polyurethane, and (10) rose bengal for UV light.We decided not to use antibiotics to coat the ETT, be-cause the indiscriminate use of antibiotics may facilitaterapid dissemination of multiresistant bacteria. Each ofthe 10 coatings showed bactericidal or bacteriostaticproperties in vitro and in animal studies. While all thesecoatings are effective to prevent bacterial colonization,we decided to select only one coating for further testing.Our selection criteria were: (1) the safety for the patientand (2) the feasibility of using the tubes in the hospital.Using PubMed and the US Food and Drug Administrationdatabase, we learned that chlorhexidine occasionallycauses immediate systemic hypersensitivity reaction [30].Carbon powder may easily detach from the coating andenter the lungs, increasing inflammatory response. Whilerose bengal is safely implemented in medicine, we suspectthat a laser source at the bedside may be difficult tomanage; furthermore, UV light use requires technicalexpertise and caution. Therefore, after thorough researchon the various coatings, we selected the ETT internallycoated with silver sulfadiazine in polyurethane. In thein-vitro study, SSD-ETT showed bactericidal propertiesagainst P. aeruginosa, preventing biofilm formation onthe ETT lumen throughout the 72-h experiments. TheSSD-ETT was also associated with decreased bacterialcolonization of the ETT, ventilator circuit, and lowerrespiratory tract in sheep mechanically ventilated for 24 hin absence of antibiotic use. No local or systemic adverse
1028
events were encountered. The present in-vitro and sheepstudies demonstrated the biological plausibility of usinga coated ETT to prevent hospital-acquired respiratory tractinfections.
However, our study presents some limitations. First,the in-vitro study assessed colonization of P. aeruginosa.No other bacteria were studied. Other bacteria might inter-act differently with the SSD coating. We chose P. aerugi-nosa because it is the most common biofilm-forming bac-terium on medical devices and it is a frequent cause ofVAP. Specifically, we used the same strain of P. aeruginosa(strain PaO1 containing the GFP-plasmid, pMRP9-1 car-benicillin resistant) used in a recent study to mimic a hu-man biofilm model [25]. In that study Singh et al. showedthe temporal sequence of a typical biofilm: adhesion oc-curs within the first 4 h, and after 24 h those bacteria formmicrocolonies. At day 3, microcolonies become confluentand cover the entire surface. By day 7, towering pillar andmushroom-shaped biofilms develop.
Second, the animal study was limited to a 24-h period.However, the main goal of this research at this stage wasto fabricate effective and safe antibacterial coatings to pre-vent bacterial colonization of the ETT, rather than decreas-ing the incidence of VAP.
In a clinical study, we tested the SSD-ETT in patientsintubated and mechanically ventilated for up to 24 h. Nobacteria were found in the ETT lumen and in the lower res-piratory tract of patients in the SSD-ETT group (Berra etal., manuscript submitted [31]). Using a different coating,Rello et al. showed in a prospective, randomized, single-blind, multicenter study that during the first 7 days of in-tubation coated ETT are associated with delayed coloniza-tion on the tube compared with non-coated ETT, and witha lower bacterial burden in tracheal aspirates [13].
Further studies should focus on developing novel an-tibacterial coatings and should evaluate in the clinical set-ting whether a decrease in bacterial colonization is associ-ated with favorable clinical endpoints.
Conclusion
The prevention of formation of a bacterial biofilm withinETT is a challenging and expanding field. We fabricatedseveral effective and durable antibacterial coatings forETT. We propose the use of ETT internally coated withsilver sulfadiazine in polyurethane in patients who areintubated and mechanically ventilated.
References
1. Vincent JL, Bihari DJ, Suter PM,Bruining HA, White J, Nicolas-Chanoin MH, Wolff M, Spencer RC,Hemmer M (1995) The prevalenceof nosocomial infection in intensivecare units in Europe. Results of theEuropean Prevalence of Infection inIntensive Care (EPIC) Study. EPICInternational Advisory Committee.Jama 274:639–644
2. National Nosocomial InfectionsSurveillance System (2004) NationalNosocomial Infections Surveillance(NNIS) System report, data summaryfrom January 1992 through June 2004.Am J Infect Control 32:470–485
3. Sottile FD, Marrie TJ, Prough DS,Hobgood CD, Gower DJ, Webb LX,Costerton JW, Gristina AG (1986)Nosocomial pulmonary infection: pos-sible etiologic significance of bacterialadhesion to endotracheal tubes. CritCare Med 14:265–270
4. Inglis TJ, Millar MR, Jones JG, Robin-son DA (1989) Tracheal tube biofilm asa source of bacterial colonization of thelung. J Clin Microbiol 27:2014–2018
6. Hartmann M, Guttmann J, Muller B,Hallmann T, Geiger K (1999) Reductionof the bacterial load by the silver-coatedendotracheal tube (SCET), a labo-ratory investigation. Technol HealthCare 7:359–370
7. Jones DS, McMeel S, Adair CG,Gorman SP (2003) Characterisation andevaluation of novel surfactant bacterialanti-adherent coatings for endotrachealtubes designed for the preventionof ventilator-associated pneumonia.J Pharm Pharmacol 55:43–52
8. Balazs DJ, Triandafillu K, Wood P,Chevolot Y, van Delden C, Harms H,Hollenstein C, Mathieu HJ (2004)Inhibition of bacterial adhesion onPVC endotracheal tubes by RF-oxygenglow discharge, sodium hydrox-ide and silver nitrate treatments.Biomaterials 25:2139–2151
9. Chaiban G, Hanna H, Dvorak T, Raad I(2005) A rapid method of impregnatingendotracheal tubes and urinary catheterswith gendine: a novel antiseptic agent.J Antimicrob Chemother 55:51–56
10. Olson ME, Harmon BG, Kollef MH(2002) Silver-coated endotrachealtubes associated with reduced bacterialburden in the lungs of mechanicallyventilated dogs. Chest 121:863–870
11. Berra L, De Marchi L, Yu ZX, La-querriere P, Baccarelli A, Kolobow T(2004) Endotracheal tubes coatedwith antiseptics decrease bacterialcolonization of the ventilator cir-cuits, lungs, and endotracheal tube.Anesthesiology 100:1446–1456
12. Berra L, Curto F, Li Bassi G, La-querriere P, Baccarelli A, Kolobow T(2006) Antibacterial-coated trachealtubes cleaned with the Mucus Shaver:a novel method to retain long-termbactericidal activity of coated trachealtubes. Intensive Care Med 32:888–893
1029
13. Rello J, Kollef M, Diaz E, Sandiu-menge A, del Castillo Y, Corbella X,Zachskorn R (2006) Reduced burdenof bacterial airway colonization witha novel silver-coated endotrachealtube in a randomized multiple-center feasibility study. Crit CareMed 34:2766–2772
14. Pacheco-Fowler V, Gaonkar T,Wyer PC, Modak S (2004) Antisepticimpregnated endotracheal tubes for theprevention of bacterial colonization.J Hosp Infect 57:170–174
15. Neuhauser MM, Weinstein RA, Ry-dman R, Danziger LH, Karam G,Quinn JP (2003) Antibiotic resistanceamong gram-negative bacilli in USintensive care units: implications forfluoroquinolone use. Jama 289:885–888
16. Archibald L, Phillips L, Monnet D,McGowan JE Jr, Tenover F, Gaynes R(1997) Antimicrobial resistance inisolates from inpatients and outpatientsin the United States: increasing impor-tance of the intensive care unit. ClinInfect Dis 24:211–215
17. Russell AD, Hugo WB (1994) Antimi-crobial activity and action of silver.Prog Med Chem 31:351–370
18. Petri WA Jr (2005) Sulfonamides,trimethoprim–sulfamethoxazole,quinolones, and agents for urinarytract infections. In: Brunton L, Lazo J,Parker K (ed) Goodman & Gilman’sThe pharmacological basis of thera-peutics, 11th edn. McGraw-Hill, NewYork, pp 1111–1127
19. Chambers HF (2006) Disinfectants, an-tiseptics, & sterilants. In: BG Katzung(ed) Basic and clinical pharmacology,10th edn. McGraw-Hill, New York,chap 50
21. Dahl TA, Midden WR, Neckers DC(1988) Comparison of photodynamicaction by Rose Bengal in Gram-positiveand Gram-negative bacteria, PhotochemPhotobiol 48:607–612
22. Berra L, De Marchi L, Pohlmann J,Kolobow T (2003) A new approachto keep the tracheal tube, ventilatorcircuit and lower respiratory tract freefrom bacterial colonization duringmechanical ventilation. AnesthesiologyAnnual Meeting Abstract Book: A417
23. Berra L, Kolobow T, De Marchi L,Mahar R, Appleton J, Corato M,Castello K, Lewandowski R (2003)Oligodynamic iontophoresis: pre-venting bacterial colonization in theventilator gas tubing, the trachealtube, the trachea, and the lungs duringmechanical ventilation. Am J Resp CritCare Med 167(7):605 (Abstract)
24. Berra L, De Marchi L, Kolobow T(2003) An internal coating with sil-ver sulfadiazine and chlorhexidinein polyurethane; or silver, platinumand carbon in polyurethane preventedbacterial colonization of the trachealtube (ETT), plus the ventilator circuit,and lower respiratory tract. ASAIO– ISAO Joint Conference 2003, Wash-ington DC, Annual meeting abstractbook
25. Singh PK, Parsek MR, Green-berg EP, Welsh MJ (2002) A com-ponent of innate immunity pre-vents bacterial biofilm development.Nature 417:552–555
26. O’Grady NP, Alexander M,Dellinger EP, Gerberding JL, Heard SO,Maki DG, Masur H, McCormick RD,Mermel LA, Pearson ML, Raad II,Randolph A, Weinstein RA (2002)Guidelines for the preventionof intravascular catheter-relatedinfections. Centers for Disease Controland Prevention. MMWR RecommRep 51:1–29
27. Johnson JR, Kuskowski MA, Wilt TJ(2006) Systematic review: antimi-crobial urinary catheters to preventcatheter-associated urinary tract infec-tion in hospitalized patients. Ann InternMed 144:116–126
28. Karchmer TB, Giannetta ET, Muto CA,Strain BA, Farr BM (2000) A random-ized crossover study of silver-coatedurinary catheters in hospitalized pa-tients. Arch Intern Med 160:3294–3298
29. Snelling CF, Inman RJ, Germann E,Boyle JC, Foley B, Kester DA,Fitzpatrick DJ, Warren RJ, Courte-manche AD (1991) Comparison ofsilver sulfadiazine 1% with chlorhex-idine digluconate 0.2% to silversulfadiazine 1% alone in the prophy-lactic topical antibacterial treatment ofburns. J Burn Care Rehabil 12:13–18
30. US-FDA Public Health Notice (1998)Potential hypersensitivity reactions tochlorhexidine-impregnated medicaldevices (ed. Burlington B) Availableat www.fda.gov/cdrh/chlorhex.html.Accessed March 29, 2008
31. Berra L, Kolobow T, Laquerriere P,Pitts B, Bramati S, Pohlmann J,Marelli C, Panzeri M, Brambillasca P,Villa F, Baccarelli A, Bouthors S,Stelfox HT, Bigatello LM, Moss J,Pesenti A (2008) Internally coated en-dotracheal tubes with silver sulfadiazinein polyurethane to prevent bacterial col-onization: a clinical trial. Intensive CareMed DOI 10.1007/s00134-008-1100-1
![Calix[4]tubes: An Approach to Functionalization](https://static.documents.page/doc/80x56/634ce6ef7b55c75a3406c83f/calix4tubes-an-approach-to-functionalization.jpg)
