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TRENDS in Microbiology Vol.9 No.1 J anuary 200134 ReviewReviewReviewReviewReview
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Review
Thien-Fah C.Mah
George A.O’Toole*
Dept of Microbiology and
Immunology,
Dartmouth Medical
School,
Hanover, NH 03755, USA.
*e-mail:
B acteria l biofilms a re formed when un icellular
organisms come together to form a community tha t is
at ta ched to a solid surface an d encas ed in an
exopolysaccharide ma trix. B iofilms ca n be m ad e up of
single or mult iple ba cteria l species. For example, it ha s
been estimat ed tha t denta l biofilms contain
>500 different bacterial ta xa1; conversely, in t he lat ter
sta ges of the disease, the prima ry bacterium in the
lungs of cystic fibrosis (CF) pa tient s is Pseudomonas
aeruginosa . It ha s been observed tha t th e resista nce ofbiofilms t o ant ibiotics is increased compared w ith w ha t
is normally seen wit h plankt onic cells. In fact, w hen
cells exist in a biofilm, th ey ca n become 10–1000 times
more resista nt t o the effects of antimicrobial a gents2–5.
It is becoming increasingly clear tha t biofilms ha ve
a n enormous impact on medicine. Biofilms can form
on many medical implant s such as catheters,
ar tificial hips and conta ct lenses a nd, owing to their
increased resista nce to ant imicrobial a gents, these
infections can often only be trea ted by remova l of the
implan t, thus increasing th e trauma to the pat ient
an d the cost of treatm ent. It ha s been estima ted tha t
biofilms a re a ssocia ted w ith 65%of nosocomialinfections 6 an d tha t t reat ment of these biofilm-based
infections costs >$1 billion an nua lly7–9.
The development of biocide resist a nce is not
underst ood, but recent st udies have used a va riety of
model syst ems to determine how a nd wh y biofilms are
so resistant to ant imicrobial a gents. As the
importa nce of biofilms in nosocomia l infections ha s
increased, much energy ha s been directed towa rds th e
study of the effects of an timicrobial a gents on th ese
surfa ce-a tt a ched communities. The key quest ion we
ask in this review is: wha t a re the mechan isms of
biofilm resista nce to a ntimicrobial compounds? Wha t
we w ill empha size is tha t t here are multiple
mechanisms, w hich va ry w ith the ba cteria present in
the biofilm a nd t he drug or biocide being a pplied.
These mechan isms include physical or chemica l
diffusion bar riers to ant imicrobial penetra tion into the
biofilm, slow growt h of the biofilm owing to nut rient
limita tion, activat ion of the genera l stress response
a nd t he emergen ce of a biofilm-specific phenoty pe. In
this review, w e will focus our a tt ention on in vi tro -
derived sing le-species biofilms, a lth ough some dua l-
species biofilm w ork will be high light ed.
Failure of the antimicrobial to penetrate the biofilm
The production of a n exopolysa ccha ride ma tr ix, or
glycocalyx, is one of the distinguishing chara cteristics
of biofilms. It h as been suggested tha t t his ma trix,
a mong other functions, prevents t he a ccess of
a ntibiotics to the ba cterial cells embedded in th e
communit y. We will highlight a few of the m ore recent
studies on t he subject of ant ibiotic diffusion t hrough a
biofilm. For a more comprehen sive review of this
subject, the reader is directed to a review by S tew a rt 10.
Eit her rea ction of the compound w ith, or sorption
to, the components of the biofilm can limit th e
tra nsport of a ntimicrobial a gents to the cells withinthe biofilm. Although ma thema tical models suggest
tha t, for many a ntibiotics, there should be no barrier
to their diffusion into a biofilm, some studies ha ve
shown a n a pparent failure of certa in an timicrobial
a gents t o penetra te th e biofilm. Chlorine, a commonly
used disinfecta nt, did not rea ch >20%of th e bulk
media’s concentra tion with in a mixed Klebsiella
pneumoniae a nd P. aeru ginosa biofilm, as mea sured
by a chlorine-detecting microelectrode11. In fact, the
penetra tion profile wa s suggestive of a subst ra te being
consumed within the ma trix. Suci et al . used infra red
spectroscopy to show t ha t t he rat e of tra nsport of the
a nt ibiotic ciprofloxacin to th e surfa ce of a colonizedsurface was reduced compared w ith tra nsport to a
sterile surface12. These authors suggested tha t t he
ciprofloxacin w as binding t o the biofilm components.
Other groups have ta ken different a pproaches t o
add ress the question of whether t he biofilm a cts as a
barrier to ant imicrobial a gents. On the one hand,
P. aeru ginosa biofilms wer e formed on one side of a
dialysis membrane a nd the a mount of piperacillin tha t
penetra ted the biofilm w as measured. Consistent with
the results discussed a bove, the P. aeru ginosa biofilm
prevented diffusion of this a ntibiotic13. On the other
hand, Staphyl ococcus epiderm id is biofilms formed in a
similar m a nner a llowed for the diffusion of rifampicin
Biofilms are communities of microorganisms attached to a surface.It has
become clear that biofilm-grown cells express properties distinct from
planktonic cells,one of which is an increased resistance to antimicrobial
agents.Recent work has indicated that slow growth and/or induction of an
rpoS -mediated stress response could contribute to biocide resistance.The
physical and/or chemical structure of exopolysaccharides or other aspects of
biofilm architecture could also confer resistance by exclusion of biocides from
the bacterial community.Finally,biofilm-grown bacteria might develop a
biofilm-specific biocide-resistant phenotype.Owing to the heterogeneous
nature of the biofilm,it is likely that there are multiple resistance mechanisms
at work within a single community.Recent research has begun to shed lighton how and why surface-attached microbial communities develop resistance
to antimicrobial agents.
Mechanisms of biofilm resistance toantimicrobial agents
Thien-Fah C.Mah and George A.O’Toole
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35ReviewReviewReviewReviewReviewReview
an d van comycin a cross the membrane14, implying tha t
th ese an tibiotics could efficiently penetr a te t his biofilm.
These results sugg est t ha t inh ibition of diffusion can not
a lwa ys explain resista nce to ant imicrobial compounds.
A difference betw een thick an d th in biofilms an d
th eir resista nce to ant ibiotics has been observed.
P enetra tion of a t hin biofilm-covered bead [a vera ge
cell densit y ~ 3.5 log colony-forming un its (cfu) cm−2
]by hyd rogen peroxide wa s observed directly, even
th ough the cells wit hin th e biofilm were more
resistant to the compound compared w ith plankt onic
cells15. B y contrast , th icker biofilms, grown on glass
slides (a vera ge cell density ~ 7.6 log cfu cm−2),
presented a barrier to th e penetra tion of hydrogen
peroxide. Interest ingly, hydr ogen peroxide wa s able
to penetra te a thick biofilm formed by a mut an t st rain
of P. aeru ginosa tha t lacked one of the ma jor cat ala se
genes, katA (Ref. 16). As cata lases a re enzymes tha t
neutr a lize hydr ogen peroxide, th is result suggested
th a t, in t hick biofilms, cells were protected from
hydrogen peroxide penetra tion by th e cata lase-media ted dest ruction of this compound.
Anderl et al . formed K. pneumoniae colony biofilms
on aga r plates with or without a ntibiotic17. By placing
a filter at the t op of the colony, essentia lly
sandw iching the colony, they w ere able to assay
directly for a ntibiotic diffusion from the a ga r plat e
thr ough the colony by performing a sta nda rd zone of
inhibition a ssa y wit h the filter. This breakth rough
study showed tha t a mpicillin wa s unable to penetra te
the biofilm a nd th a t t he production of the a mpicillin-
degrading enzyme β-lacta ma se wa s responsible for
this phenomenon, as t he ampicillin wa s able to
penetra te a biofilm formed by a β-lactama se muta nt.Surprisingly, th e β-lactama se mutant s grown in a
biofilm were still resistan t t o ampicillin, suggesting
tha t other mechanisms contr ibute to the resista nce of
these cells. Furt hermore, ciprofloxacin w as able t o
penetrat e the biofilm, yet, as w as the case with
a mpicillin, it w a s una ble to kill the biofilm bacteria 17.
This simple method a llowed for t he differentiat ion
between tra nsport effects a nd other mecha nisms and
thu s provides a powerful tool for the further a na lysis
of the molecular mechanism of biofilm resista nce to
an timicrobial agents.
From these studies, an d others, it is clear t ha t th e
exopolysa ccha ride ma trix (or other components ofbiofilms) does not form a n impenetra ble ba rrier to th e
diffusion of a ntimicrobial a gents, a nd other
mechan isms mu st be in place to promote biofilm cell
surviva l. However, for certa in compounds, the
exopolysaccha ride ma trix does represent a n initia l
barrier tha t can delay penetra tion of the
a nt imicrobial a gent. The experiment s described
above strongly suggest tha t multiple mechanisms a re
required for overa ll a ntim icrobial resista nce.
Slow growth and the stress response
When a ba cterial cell cultur e becomes sta rved for a
part icular n utrient, it slows its growt h. Tra nsition
from exponentia l to slow or no growt h is genera lly
accompanied by a n increase in resistance to
antibiotics 18,19. Slow growth of the bacteria ha s been
observed in mat ure biofilms20,21. B eca use cells
growing in biofilms a re expected to experience some
form of nutrient limitat ion, it ha s been suggested tha t
th is physiologica l cha nge can a ccount for the
resista nce of biofilms to a nt imicrobial a gents.B y paying close at tention to the growt h phase of
plankt onic cells an d biofilm cells, recent stud ies ha ve
been able t o exa mine specifically t he contribution of a
slow growth rat e to biofilm cell survival a gainst
an tibiotics. Gilbert a nd collea gues examined
growt h-ra te-relat ed effects und er controlled growt h
condit ions for pla nkt onic cultures a nd biofilms of
P. aeru ginosa , Escher ichi a coli a nd
S. epidermi dis 22–24. They ma de the general
observation tha t t he sensitivities of both the
plankt onic a nd biofilm cells to either tobra mycin or
ciprofloxacin increased with increa sing growth ra te,
thus supporting th e suggestion tha t t he slow growt hra te of biofilm cells protects th e cells from
an timicrobial a ction. For P. aeru ginosa at slow
growth r at es, both the plankt onic and int act biofilm
cells were equa lly resista nt t o ciprofloxacin. However,
as t he growth ra te wa s increased, the planktonic cells
beca me more susceptible to ciprofloxacin t ha n t he
biofilm cells. This result support s th e idea t ha t some
other propert y of the biofilm, and not just growin g
slowly, w as importa nt for t he observed recalcitrance
of biofilms to a nt imicrobial tr eat ment 22. The sa me
group reached a simila r conclusion w hile working
wi th Bu rk holderia cepacia . Desai et al . compared the
resista nce of plankt onic a nd biofilm cells at differentsta ges during exponential growth up to the entry into
stat ionary phase25. They found tha t resista nce
increased a s t he planktonic cultures a nd t he biofilm
cells approa ched st at iona ry pha se. The ma ximal
resista nce of both cultures occurred in sta tiona ry
pha se wh ere the biofilm cells were 15-times m ore
resista nt t ha n the plan ktonic cells. These results
suggested that some determinant other tha n growt h
ra te is responsible for a certa in level of resista nce, and
slow growth a dds a dditiona l protection. This
determinant could be relat ed to the fact t ha t cell
density increases dur ing this la te sta ge of exponential
growth (see below). Other studies ha ve suggested tha tmechan isms differ for different a nt ibiotics. For
example, although the slow growth ra te in a
P. aeru ginosa biofilm seemed t o account for biofilm
resista nce to tet ra cycline, it did not seem to a ffect
resistance to t obra mycin26.
Heterogeneity
The experiment a l conditions resulting in t he tight
cont rol of growt h described in the stud ies
summ a rized above a llow ed investiga tors to focus on
th e effect of a specific growt h ra te on bacteria l
susceptibility t o an timicrobial a gents. H owever, w hen
thinking a bout biofilms, a logical a ssumption is t ha t
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a ny given cell with in th e biofilm will experience a
slightly different environment compa red with oth er
cells within th e same biofilm, an d thus be growing a t
a different rat e. Gr adients of nutrients, wa ste
products a nd signa ling factors form t o allow for t hisheterogeneity w ithin th e biofilm.
Recent a dvances in t echnology ha ve resulted in the
ability t o visualize the heterogeneity with in a biofilm.
A staining method utilizing a cridine oran ge wa s
employed to identify regions of biofilms th a t conta in
ra pidly or slowly growing cells based on their relat ive
RNA–DNA cont ent 21. The method w a s first used on
bact erial colonies and t he regions of the colonies th a t
tur ned oran ge (high rela tive RNA cont ent) were
correlated to fast growt h ra tes, whereas regions tha t
were sta ined yellow/green (low relat ive RNA cont ent)
represented slowly gr owing cells. When th is method
wa s used on seven-da y-old biofilms, orang e ma rkedth e biofilm–bulk-liqu id inter fa ce a nd yellow /green
ma rked t he center of th e biofilm (Fig. 1). This
heterogeneity w ithin biofilms ha s also been show n for
protein synthesis an d respira tory activity, whereas
DNA content rema ined relatively constant
th roughout th e biofilm27,28.
There is a lso evidence for gra dients of
physiological a ctivity in response to a nt imicrobial
trea tment. F or example, the patt ern of respira tory
a ctivity of a K. pneumoniae biofilm in response t o
monochloram ine (a n oxidat ively act ive biocide)
trea tment sh owed tha t cells closest to the
biofilm–bulk-liquid int erface lost a ctivity first 29
(Fig. 2). Similar ly, when biofilm cells were trea ted
wit h th e ant ibiotic fleroxocin, cell elongat ion wa s
observed a nd w a s most extreme in cells loca ted close
to th e exposed sid e of th e biofilm30. These st udies
reveal t ha t t he response to ant imicrobial a gents can
great ly vary , depending on the locat ion of a part icular
cell with in a biofilm communit y.
General stress response
Recently, it has been suggested tha t t he slow growt h
ra te of some cells wit hin th e biofilm is not owin g to
nutrient limitat ion per se , but to a general str ess
response initiated by growth with in a biofilm31. This
idea is a n a tt ra ctive possibility because t he stress
response results in physiological changes tha t a ct to
protect the cell from va rious environment a l stresses.
Thus, th e cells are protected from the detr imenta l
effects of hea t sh ock, cold shock, cha nges in pH a nd
ma ny chemical a gents32. The centra l regulat or of thisresponse is the alterna te σ factor, RpoS, originally
thought t o be expressed only in sta tionary pha se32.
However, recent st udies suggest t ha t RpoS is induced
by high cell density an d tha t cells growing a t t hese
high densities seem t o have undergone the general
str ess response, as judged by t he production of
treha lose (an osmoprotectant ) an d cata lase33. As cells
in a biofilm experience high cell density, it is logica l to
propose tha t t hese cells would express RpoS.
Accordingly, it ha s been shown by RT-P CR t ha t
rpoS mRNA is present in sputum from CF patients
with chronic P. aeru ginosa biofilm infections34.
Another link betw een RpoS a nd biofilms wa srecently identified: E. coli cells tha t lack rpoS a re
una ble to form n ormal biofilms wh ereas plan ktonic
cells are a pparently una ffected by the a bsence of this
σ factor35. In P. aeru ginosa , it ha s been suggested tha t
an additional σ fact or, AlgT, a cts in concert wit h RpoS
to cont rol th e stress response31,34. Cochra n et al . found
tha t th in biofilms formed by null muta nts of rpoS a nd
algT on algina te gel beads w ere susceptible to
hyd rogen peroxide but not to monochlora mine36.
However, when these mut an ts formed th ick biofilms on
glass slides, they were as resista nt t o both oxidat ive
biocides a s th e wild-ty pe cells. Thus, a lthough th ere is
some evidence to suggest t ha t rpoS a nd algT have a
Fig. 1. Physiological heterogeneity in biofilms. The spatial pattern of growth rate within a Klebsiella pneumoniae biofilm, as judged by
acridine orange staining. In this figure, areas of red–orange staining
correspond to a high relative RNA content and thus rapid growth. Cells
staining yellow/green have low relative RNA content and a slower
growth rate. There are clearly distinct regions of faster and slower
growth throughout the biofilm. The bottom of the image is the portion
of the biofilm attached to the substratum and the top of the image is the
portion of the biofilm exposed to the bulk medium. Reproduced, with
permission, from Ref.21.
Fig. 2. Susceptibility to biocide treatment. A two-species biofilm treatedwith the oxidatively active biocide monochloramine. This figure illustrates
that there is heterogeneity within the biofilm in terms of the response of
individual cells to biocide treatment. Areas of red–orange staining
correspond to respiratory activity. Green cells have no respiratory activity.
Yellow regions represent a mixture of respiring and non-respiring
bacteria. The bottom of the image is the portion of the biofilm attached to
the substratum and the top of the image is the portion of the biofilm
exposed to the bulk medium. Reproduced, with permission, from Ref. 29.
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role in biofilm resist a nce to oxida tive biocides, it is clear
tha t other factors must contribute to this resista nce.
Quorum sensing
The role of quorum s ensing in biocide resist a nce is not
yet clear. P revious w ork by Da vies and colleagues
showed that a mutant in the l asR–lasI quorum-sensing
system in P. aeru ginosa wa s una ble to form a biofilmwit h norma l ar chitecture37. Moreover, these aut hors
presented data showing tha t lasI muta nt biofilms were
abnormally sensitive to treatment with SDS , although
the question of wh ether these muta nt biofilms had
a ltered antibiotic resistan ce wa s not addressed37.
However, a recent st udy by B rooun a nd co-workers
showed tha t mut an ts defective in quorum sensing were
una ffected in their resista nce to detergents an d
antibiotics26. Furt her complica ting t he interpretat ion
of these studies is a report suggesting a role for RpoS in
regulation of quorum sensing38. Addit ional
experimenta tion is required t o elucidat e the role (direct
or indirect) of quorum s ensing in biocide resista nce.
Induction of a biofilm phenotype
Thus fa r, th e mecha nisms discussed ha ve been ba sed
on general st ra tegies to slow t he effect of
a nt imicrobial a gents on cells in th e biofilm. An
emerging idea in th e field is th a t a biofilm-specific
phenotype is induced in a subpopulation of the
community tha t results in t he expression of a ctive
mecha nisms to combat the detrimenta l effects of
an timicrobial agents 15,39–41.
When cells at ta ch to a surfa ce, th ey will express a
general biofilm phenotype and w ork has begun t o try
to identify genes tha t a re activat ed or repressed inbiofilms compa red w ith plankt onic cells42.
Furth ermore, it is possible tha t a ll or just a subset of
th ese biofilm cells could express increased resista nce
to a ntimicrobial a gents. This resistan t phenotype
might be induced by nut rient limitat ion, certa in types
of stress, high cell density or a combinat ion of these
phenomena. As summa rized below, recent w ork has
focused on th e identificat ion of genes th a t could
cont ribute to this increa sed-resista nce phenotype.
Multidru g efflux pumps can extr ude chemica lly
unrelated a ntimicrobial a gents from the cell. In
E. col i , upregulat ion of the mar operon results in a
mult idrug-resista nt phenotype. The efflux pump
th ought t o be responsible for this resist a nce is AcrAB .
To address t he question of whether t his known
multid rug-resista nce system is involved in biofilm
resista nce to an timicrobial agent s, expression of mar
wa s monitored in batch, chemosta t a nd biofilm
cultures by lacZ fusion 41. Overall, the results did not
support the idea tha t the mar operon is upregula ted
in biofilms, a s t he level of mar w a s lower in biofilmscompa red wit h th e level seen in equiva lent
sta tionary-phase culture grown in bat ch.
Furth ermore, this sa me group made use of mar- a nd
acrAB -deleted stra ins to determine if the resista nce of
E. col i to ciprofloxacin w a s a ffected by loss of these
loci40. Loss of mar a nd acrAB did not ad versely affect
the E. col i biofilms, but const itut ive expression of
acrAB did provide a certa in level of protection aga inst
ciprofloxacin. Although t hese results suggest th a t
upregula tion of the mar operon specifica lly does not
a ccount for E. coli biofilm resista nce to ant imicrobial
a gents , they do not discount th e possibility of oth er
multid rug-resista nce pumps being induced inresponse to life in a biofilm.
There a re th ree known m ultidru g-efflux pumps in
P. aeru ginosa an d there are several other putat ive
pumps tha t ha ve been identified by the P. aeru ginosa
genome project. One study h a s suggested th e
importa nce of one of these pumps in the resista nce to
th e a ntibiotic ofloxa cin26. Using stra ins of
P. aeru ginosa tha t either lacked or overexpressed t he
MexAB –OprM pump, it wa s shown tha t, a t low
concentra tions of ofloxacin, biofilms lacking t he pump
were more susceptible to this drug t ha n biofilms th a t
overexpressed th e pump. However, for a d ifferent
quinolone, ciprofloxacin, th ere w a s no difference.Therefore, as wa s the case w ith t he E. col i studies, the
quest ion of wh ether ind uction of pumps is one of the
key alt era tions conferring resist a nce to biofilm cells
aw aits further experimenta tion.
Another resistance mecha nism tha t can be
induced in biofilm cells is t he a lterat ion of the
membra ne-protein composition in r esponse to
a ntimicrobial a gents. This chan ge could result in
decreas ed permeabilit y of th e cell to th ese
compounds. Muta tions in ompB (a regulat or of the
Fig. 3. Drug resistance in
biofilms. A schematic of
mechanisms that can
contribute to the
resistance of biofi lm-
grown bacteria to
antimicrobial agents. The
extracellular
polysaccharide is
represented in yellow andthe bacteria as blue ovals.
Biofilms are marked by
their heterogeneity and
this heterogeneity can
include gradients of
nutrients, waste products
and oxygen (illustrated by
colored starbursts).
Mechanisms of resistance
in the biofilm include
increased cell density and
physical exclusion of the
antibiotic. The individual
bacteria in a biofilm can
also undergo
physiological changes
that improve resistance to
biocides. Various authors
have speculated that the
following changes can
occur in biofilm-grown
bacteria: (1) induction of
the general stress
response (an rpoS -
dependent process in
Gram-negative bacteria);
(2) increasing expression
of multiple drug
resistance (MDR) pumps;
(3)activating quorum-
sensing systems; and
(4)changing profiles of
outer membrane proteins(OMP).
TRENDS in Microbiology
+ +
+ +
Antibiotic
concentrationNutrient andoxygenconcentration
Quorum
sensing
RpoS
MDR pumps
OMP
Wha t other factors ar e importa nt for
a ntim icrobial resist a nce in multi-species
biofilms?
• Wha t genes ar e induced in biofilm cells tha t
allow for increased resista nce to antimicrobial
agents?
• Wha t signa ls are involved in rpoS regulation
in biofilms?
• Are multidr ug efflux pumps importa nt for
biofilm resistance to an timicrobial a gents?
• Wha t is th e role of quorum sensing in t he
biocide resist a nce developed by biofilms?
Questions for future research
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genes encoding t he outer membra ne porin proteins
OmpF and OmpC) and in ompF increa sed the
resista nce of E. col i t o a β-lacta m an tibiotic43.
Mutant s tha t la ck OmpF ha ve been shown to be more
resista nt t o chloramphenicol and t etra cycline44.
Furt hermore, in sta rving cells, the relat ive
proport ions of the ma jor E. col i porins OmpC a nd
OmpF w ere altered, fa voring t he expression of thesma ller porin, OmpC (Ref. 45). The a bove result s
support the suggestion th at a ltering porin expression
a ffects the intrinsic resista nce of bacteria t o
ant imicrobial agents. Recently, it w as shown tha t t he
expression of ompC an d thr ee other osmotica lly
regulat ed genes wa s increased in biofilm bacteria
compared wit h pla nktonic cells46. These da ta
suggested tha t ba cteria in a biofilm a re indeed living
in a n environment of increas ed osmotic st ress. Thus,
the environmenta l conditions wit hin th e biofilm can
lead to a lterat ions w ithin th e cell envelope tha t
protect the ba cteria from the detrimenta l affects of
antimicrobial agents.
Conclusion
There is no one a nsw er to the quest ion of why an d how
ba cteria gr owing in a biofilm develop increased
resistan ce to an timicrobial ag ents. We have seen tha t
there a re man y possible mecha nisms tha t a ccount for
bacterial resistan ce to ant imicrobial compounds
(summa rized in Fig. 3). Depending on th e bacterial
complement of the biofilm, and the a ntimicrobial a gent
used to trea t t he biofilm, different m echa nisms w ill
account for r esistance t o the an timicrobial compound.
Furt hermore, the environmenta l heterogeneity t ha texists w ithin a biofilm might promote the format ion of
a h eterogeneous populat ion of cells, such tha t different
levels of resist a nce ca n be expressed thr oughout th e
commu nity . For example, the cells closest t o the
liquid–biofilm interface might be protected to a sma ll
degree by t he exopolysaccharide ma trix a nd by
enzymes th at inactivat e certa in a ntimicrobial agents.
The cells in an int ermedia te position might be growing
slowly a nd could also be protected by the outerm ost
lay er of cells. Fina lly, a nother s ub-popula tion of cells
might express a biofilm-specific resista nce phenotype
induced by th e particular environmental fa ctors
influencing these cells. It is clear tha t a dditionalstudies must be performed t o further elucidat e how
an d wh y ba cteria growing in complex surface-at ta ched
commu nities can protect t hemselves from the insults of
an timicrobial agent s.
Acknowledgements
We wish to thank Phil
Stewart for permission to
use Figs 1 and 2. This
work was supported by agrant from Microbia, Inc.
and The Pew Charitable
Trusts (to G.A.O.). G.A.O.
is a Pew Scholar in the
Biomedical Sciences.
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Since their recognition a s the th ird doma in of life,
Archaea, par ticularly t hose tha t t hrive in extreme
environments , have been the focus of a g rea t dea l of
recent resea rch, including complete genome sequence
determination1. However, th e viruses of these
organ isms a re poorly underst ood. A survey of the
extra -chromosoma l element s of the extremely
th ermophilic and a cidophilic a rcha eon Sulfolobus ha s
revealed t he presence of many novel viruses, a nd both
conjugat ive a nd cryptic plasm ids2,3. The vir uses of
Sulfolobus ha ve been a ssigned t o four novelfamilies – Fuselloviridae (viruses SS V1, SS V2 an d
SSV3)3–5, Rudiviridae (SIR V1 an d SIRV2)6,
Lipothrixviridae (SIFV)7 and G ut tav i ridae
(SNDV)8 – on the ba sis of their unique morphology.
Morphology
The str ucture of typical virus pa rticles from each of
th e four fa milies is shown in F ig. 1. The flexible
filament ous virions of the lipothrixvirus SI FV
(2000 × 24 nm) cont a in a nucleosome-like core of
linear DNA wound as a superhelix around a zipper-
like arr a y of 80-kDa protein subunit s. The core is
covered by a lipid envelope (Fig. 1a). By cont ra st, t he
stiff rod-sha ped virions of rudiviruses
(830–900 × 23 nm) do not possess an envelope. In t he
Rudivirida e, the t ube-like superhelix formed by
linear DNA an d a single 15.8-kDa DNA-binding
protein is closed a t its en ds by ‘plugs’ to w hich ta il
filament s are a tt a ched (Fig. 1b). The spindle-sha ped
fusellovirus v irions (100 × 60 nm) have a core of
positively supercoiled circular D NA associated w ith aDNA-binding protein, pa ckaged in a hyd rophobic
protein envelope, wh ich has a short t a il (Fig. 1c).
Fina lly, virions of the gutt avirus SND V
(100–185 × 70–95 nm) ta ke the form of droplets,
wh ich, on th eir pointed end, ca rry a dense ‘beard ’ of
long t hin filaments (Fig. 1d).
Natural hosts and geographical distribution
Sulfolobus viruses appear to be ubiquit ous in acidic
hot-spring environment s. Fu selloviruses ha ve been
found in Sulfolobus stra ins isolated from solfata r ic
fields in J a pan , Icelan d an d North America. The
na tura l carriers of rudiviruses a nd thelipoth rixvirus SIFV are Sulfolobus isola tes from
diverse locat ions in I celan d, a lthough viruses of
similar morphology ha ve been observed in sam ples
from North America. The gutt a virus SND V has been
found in a Sulfolobus isola te from a field sample
from New Zeala nd.
Virus–host relationships
None of the Sulfolobus viruses is lytic: th e
fuselloviruses a re temperate, a nd th e others a re
present in th eir hosts in a more-or-less-sta ble ca rrier
sta te. This str a tegy could help th e viruses escape
prolonged d irect exposure t o the low pH (1–3) and
Viruses of the extremely thermophilic
archaeonSulfolobus David Prangishvili,Kenneth Stedman and Wolfram Zillig
Viruses of Sulfolobus are highly unusual in their morphology,and genome
structure and sequence.Certain characteristics of the replication strategies of
these viruses and the virus–host interactions suggest relationships with
eukaryal and bacterial viruses,and the primeval existence of common
ancestors.Moreover,studying these viruses led to the discovery of archaeal
promoters and has provided tools for the development of the molecular
genetics of these organisms.TheSulfolobus viruses contain unique
regulatory features and structures that undoubtedly hold surprises forresearchers in the future.
David Prangishvili*
Universität Regensburg,
Lehrstuhl für
Mikrobiologie –
Archaeenzentrum,
Universitätsstraße 31,
93053, Regensburg,
Germany.
*e-mail:
david.prangishvili@
biologie.uni-r.de
Kenneth Stedman
Thermal Biology Institute,
Montana State University,
Bozeman,
MT 59717, USA.
Wolfram Zillig
Max-Planck-Institut für
Biochemie,
82152 Martinsried,
Germany.