, 374 (2006); 311
Vanessa M. D'Costa,
Sampling the Antibiotic Resistome
www.sciencemag.org (this information is current as of October 1, 2007 ):
The following resources related to this article are available online at
version of this article at:
including high-resolution figures, can be found in the online Updated information and services,
can be found at: Supporting Online Material
can be related to this articleA list of selected additional articles on the Science Web sites
, 16 of which can be accessed for free: cites 22 articles This article
47 article(s) on the ISI Web of Science. cited by This article has been
6 articles hosted by HighWire Press; see: cited by This article has been
: subject collections This article appears in the following
in whole or in part can be found at: this article
permission to reproduce of this article or about obtaining reprintsInformation about obtaining
registered trademark of AAAS.
2006 by the American Association for the Advancement of Science; all rights reserved. The title
Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
on October 1, 2007
with ORP), or that south polarity is character-
istic of a particular species that occurs at a
The coexistence of magnetotactic bacteria
with north and south polarity in the same
chemical environment contradicts the current
accepted model of magnetotaxis, which states
that all magnetotactic bacteria in the Northern
Hemisphere swim north (downward in situ)
when exposed to oxidized conditions to reach
their preferred microaerobic or anaerobic
habitat. We observed that barbells with south
polarity and cocci with north polarity coexist
in microaerobic conditions in the water col-
umn. On the basis of this distributional pat-
tern, south polarity is clearly not used to
direct the barbell upward in the water column
toward higher oxygen levels. The current
model does not, therefore, provide any expla-
nation that can account for the existence of
This model implicitly assumes that polar-
ity observed in the laboratory under atmo-
spheric oxygen levels is equivalent to polarity
in situ. Our results suggest that this assump-
tion might be incorrect. Although the benefit
of north polarity in situ is clear for micro-
aerophilic magnetotactic bacteria, south po-
larity would have a clearly deleterious effect
by directing the bacteria away from their pre-
ferred chemical environment. There are rea-
sons to believe the behavior of magneotactic
bacteria in situ could differ from behavior
in the laboratory. Magnetotactic bacteria at
the chemocline of a stratified water column
rarely, if ever, experience atmospheric oxy-
gen levels like those in the standard labora-
tory assay for polarity. They also experience
chemical gradients (particularly of iron and
sulfur species) not present in a drop of water
exposed to air in the laboratory assay. It is
also possible that bacteria with north and
south polarity possess different chemo- or
redox-sensors that have opposite responses
to chemical concentrations out of the range
they typically experience. On the basis of
these results, new models are clearly needed
to explain the adaptive significance of mag-
netotaxis by magnetotactic bacteria in the
References and Notes
1. R. P. Blakemore, Annu. Rev. Microbiol. 36, 217 (1982).
2. R. P. Blakemore, R. B. Frankel, A. J. Kalmijn, Nature 286,
3. J. L. Kirschvink, J. Exp. Biol. 86, 345 (1980).
4. S. Spring et al., Appl. Environ. Microbiol. 59, 2397
5. S. Spring, R. Amann, W. Ludwig, K.-H. Schleifer,
N. Petersen, Syst. Appl. Microbiol. 15, 116 (1992).
6. T. Sakaguchi, A. Arakaki, T. Matsunaga, Int. J. Syst. Evol.
Microbiol. 52, 215 (2002).
7. H. Petermann, U. Bleil, Earth Planet. Sci. Lett. 117, 223
8. J. F. Stolz, S.-B. R. Chang, J. L. Kirschvink, Nature 321,
9. S. L. Simmons, S. M. Sievert, R. B. Frankel, D. A.
Bazylinski, K. J. Edwards, Appl. Environ. Microbiol. 70,
10. D. A. Bazylinski et al., Appl. Environ. Microbiol. 61, 3232
11. S. L. Simmons, K. J. Edwards, manuscript in preparation.
12. R. B. Frankel, D. A. Bazylinski, M. S. Johnson, B. L. Taylor,
Biophys. J. 73, 994 (1997).
13. R. P. Blakemore, D. Maratea, R. S. Wolfe, J. Bacteriol.
140, 720 (1979).
14. A. M. Spormann, R. S. Wolfe, FEMS Microbiol. Lett. 22,
15. S. L. Simmons, K. J. Edwards, unpublished observations.
16. R. P. Blakemore, Science 190, 377 (1975).
17. T. T. Moench, W. A. Konetzka, Arch. Microbiol. 119, 203
18. Materials and methods are available as supporting
material on Science Online.
19. T. J. Lie, M. L. Clawson, W. Godchaux, E. R. Leadbetter,
Appl. Environ. Microbiol. 65, 3328 (1999).
20. R. Kawaguchi et al., FEMS Microbiol. Lett. 126, 277
21. E. F. DeLong, R. B. Frankel, D. A. Bazylinski, Science 259,
22. A. J. Kalmijn, R. P. Blakemore, in Animal Migration,
Navigation, and Homing, K. Schmidt-Koenig, W. Keeton,
Eds. (Springer-Verlag, Berlin, 1978), pp. 354-355.
23. We thank P. Canovas and O. Rafie for field and lab
assistance; R. Frankel, E. Webb, and S. Sievert for input
on the manuscript; and K. Canter for use of a
magnetometer. S.L.S. was partially supported by a
National Defense Science and Engineering Graduate
Fellowship. This work was partially funded by grants to
S.L.S. and K.J.E. from the WHOI Reinhart Coastal Research
Center, the WHOI Ocean Venture Fund, and the WHOI
Ocean Life Institute. D.A.B. is supported by National Science
Foundation grant EAR-0311950. GenBank sequence
accession numbers for the sequences reported in this study
are as follows. Group 1: BL_G6, DQ322653; BL_H12,
DQ322654; BL_H1, DQ322655; BL_H4, DQ322662,
BL_H5, DQ322656; BL_C10, DQ322657; BL_C5,
DQ322658. Group 2, south-seeking bacterium: BL_C2c,
DQ322659; BL_E6c, DQ322660; BL_G11c, DQ322661.
Supporting Online Material
Materials and Methods
21 November 2005; accepted 20 December 2005
Sampling the Antibiotic Resistome
Vanessa M. D’Costa,1Katherine M. McGrann,1Donald W. Hughes,2Gerard D. Wright1*
Microbial resistance to antibiotics currently spans all known classes of natural and synthetic
compounds. It has not only hindered our treatment of infections but also dramatically reshaped
drug discovery, yet its origins have not been systematically studied. Soil-dwelling bacteria
produce and encounter a myriad of antibiotics, evolving corresponding sensing and evading
strategies. They are a reservoir of resistance determinants that can be mobilized into the
microbial community. Study of this reservoir could provide an early warning system for
future clinically relevant antibiotic resistance mechanisms.
resistance elements for self-protection that are
often clustered in antibiotic biosynthetic operons
(2, 3). Genes orthologous to these have been
identified on mobile genetic elements in resistant
pathogens in clinical settings. It has been sug-
ost clinically relevant antibiotics orig-
inate from soil-dwelling actinomy-
cetes (1). Antibiotic producers harbor
gested that aminoglycoside-modifying kinases
(4) and the alternate peptidoglycan biosynthetic
machinery that confers resistance to vancomy-
cin (5) probably originated in soil-dwelling anti-
The presence of antibiotics in the environ-
ment has promoted the acquisition or indepen-
dent evolution of highly specific resistance
elements in the absence of innate antibiotic
production Esuch as vancomycin resistance in
Streptomyces coelicolor, Paenibacillus, and
Rhodococcus (6, 7)^. The soil could thus serve
as an underrecognized reservoir for resistance
that has already emerged or has the potential
to emerge in clinically important bacteria.
Consequently, an understanding of resistance
determinants present in the soil—the soil
resistome—will provide information not only
about antibiotic resistance frequencies but also
about new mechanisms that may emerge as
We isolated a morphologically diverse col-
lection of spore-forming bacteria from soil
samples originating from diverse locations
(urban, agricultural, and forest). Strains that
resembled actinomycetes both morphologically
and microscopically were serially subcultured
to apparent homogeneity. Amplification and
sequencing of 16S ribosomal DNA from a
subset of strains indicated that they belonged
to the actinomycete genus Streptomyces, whose
species synthesize over half of all known
antibiotics (1). We constructed a library of
480 strains that was subsequently screened
against 21 antibiotics, including natural pro-
ducts (such as vancomycin and erythromycin),
their semisynthetic derivatives (such as mino-
cycline and cephalexin), and completely syn-
thetic molecules (such as ciprofloxacin and
linezolid). The antibiotics encompassed all
major bacterial targets (8) and included drugs
1Antimicrobial Research Centre, Department of Biochemistry
and Biomedical Sciences,2Department of Chemistry, McMaster
University, Ontario, Canada, L8N 3Z5.
*To whom correspondence should be addressed. E-mail:
20 JANUARY 2006VOL 311SCIENCEwww.sciencemag.org
on October 1, 2007
that have been on the market for decades as
well as several that have only recently been
clinically approved (such as telithromycin and
The screen was conducted at high antibiotic
concentrations (Fig. 1), and strains of interest
were analyzed by determination of the minimal
inhibitory concentration (MIC). A subset of
isolates was characterized on the basis of mode
of resistance in order to distinguish resistance
arising from antibiotic alteration or modification
from that arising from nondestructive mech-
anisms (such as efflux, altered target, or trans-
port) (Table 1).
Without exception, every strain in the
library was found to be multi-drug resistant to
seven or eight antibiotics on average, with two
strains being resistant to 15 of 21 drugs (Fig.
1B). Reproducible resistance to most of the
antibiotics, regardless of origin, was observed,
and almost 200 different resistance profiles
were seen (Fig. 1, A and C), exemplifying the
immense genetic and phenotypic diversity of
the collection of bacteria.
Several antibiotics, including the synthetic
dihydrofolate reductase (DHFR) inhibitor tri-
methoprim and the new lipopeptide daptomy-
cin, were almost universally ineffective against
the library. The genomes of S. coelicolor
and S. avermitilis do not contain annotated
DHFR genes, which is consistent with insen-
sitivity to trimethoprim (9). However, exten-
sive daptomycin resistance was not anticipated.
Recently approved by the Food and Drug
Administration (FDA), daptomycin is high-
ly active against Gram-positive bacteria, in-
cluding multi-drug–resistant pathogens (10, 11).
A member of a large antibiotic class com-
monly produced by actinomycetes, daptomy-
cin is thought to act by insertion into the
bacterial cell membrane in a Ca2þ-dependent
manner (12, 13).
Eighty percent of the resistant strains assayed
inactivated daptomycin after 48 hours of cell
growth, while the remaining strains retained ac-
tive antibiotic in the culture media (Table 1).
This finding is notable not only because it is
only the second documented occurrence of
daptomycin inactivation (14) but because of its
unprecedented high frequency. Furthermore, it
suggests that there are multiple mechanisms of
daptomycin resistance in soil organisms.
We uncovered a wealth of inactivating en-
zymes produced by soil bacteria. Of the 11
antibiotics screened, bacterial isolates were
detected that putatively metabolized 6 drugs
(Table 1), including rifampicin and Synercid.
Rifampicin, a semisynthetic derivative of a
natural Amycolatopsis mediterranei product, is
central to the treatment of mycobacterial in-
fections. Forty percent of resistant isolates were
capable of inactivating the drug, which is
intriguing because clinically, the most prevalent
mechanism of rifampin resistance is through
point mutations in the target: RNA polymer-
ase_s b subunit.
Synercid, which was FDA-approved in
1999 for the treatment of drug-resistant bacte-
remia, is a combination of two semisynthetic
derivatives of Streptomyces metabolites, each
with a distinct mode of action. Eighteen percent
of resistant isolates tested were able to detoxify
both antibiotics. These findings collectively re-
inforce the importance of enzymatic antibiotic
inactivation as a means of resistance (4).
The screen yielded five strains that were
highly resistant to the glycopeptide vancomycin
(MICs of 128 to 256 mg/ml). Resistance in both
clinically significant and glycopeptide-producing
Fig. 1. Antibiotic resistance profiling of 480 soil-derived bacterial isolates. (A) Schematic diagram
illustrating the phenotypic density and diversity of resistance profiles. The central circle of 191 black
dots represents different resistance profiles, where a line connecting the profile to the antibiotic
indicates resistance. (B) Resistance spectrum of soil isolates. Strains were individually screened from
spores on solid Streptomyces isolation media (SIM) against 21 antibiotics at 20 mg of antibiotic per ml of
medium (mg/ml). Resistance was defined as reproducible growth in the presence of antibiotic. (C) Resistance
levels against each antibiotic of interest.
www.sciencemag.orgSCIENCE VOL 311 20 JANUARY 2006
on October 1, 2007
bacteria is the result of the biosynthesis of an
altered peptidoglycan terminating in D-alanine-
D-lactate rather than D-alanine-D-alanine, result-
ing in a poor binding affinity to vancomycin.
This mechanism is encoded by a cluster of three
genes, vanH-vanA-vanX, which can be readily
identified by polymerase chain reaction analysis
in resistant Streptomyces (5). Using this strategy,
the cluster was amplified in 80% of resistant
strains (fig. S1). The outlying strain AA#4 ap-
peared to be resistant by a nondestructive
mechanism and displayed a distinct glycopeptide
resistance profile. Although the vanHAX strains
demonstrated vancomycin resistance (MICs of
128 to 256 mg/ml) but sensitivity to the lipo-
glycopeptide teicoplanin (MICs of 1 to 4 mg/ml),
AA#4 was resistant to both (MICs of vancomy-
cin and teicoplanin of 256 mg/ml).
Synthetic fluoroquinolones target DNA
gyrase A (GyrA)–dependent supercoiling and
topoisomerase IV–dependent decatenation of
bacterial DNA, inhibiting DNA replication
and segregation (15, 16). Clinical resistance oc-
curs primarily through point mutations in the
N-terminal region of gyrA, termed the quinolone
resistance–determining region (QRDR) (17);
however, resistance has also been documented
through antibiotic efflux (17) and plasmid-
mediated protection of DNA gyrase (18).
Despite a lack of known prior exposure to
fluoroquinolones or bacterially synthesized
analogs, 11% of strains demonstrated intrinsic
resistance to ciprofloxacin (Fig. 1C, MICs of 6
to 128 mg/ml). Of the 52 resistant strains, none
eliminated fluoroquinolone antibacterial activi-
ty, indicating that enzymatic inactivation was
unlikely (Table 1). To investigate the possibility
of QRDR mutation, this region was cloned and
sequenced from 38 resistant isolates (Fig. 2).
Eleven different amino acid substitutions were
identified at nine QRDR locations in 24% of
strains sequenced. These included locations
commonly associated with clinical ciprofloxacin
resistance (such as Ser83and Asp87, using the
Escherichia coli numbering system), as well as
novel sites within the QRDR (such as Met100
and Ser110). Among these strains, the isolate
with the highest MIC displayed a mutation at a
novel location (Ser110). The high incidence of
mutations in the absence of obvious environ-
mental selective pressures is consistent with
Fig. 2. ProteinsequencealignmentoftheQRDRofciprofloxacin-resistantstrainsexhibitingmutations.
A 266–base pair region of gyrA was amplified in resistant strains and sequenced. Mutations are labeled
in orange, and residues are numbered according to the E. coli system. Black sites are completely
conserved among the 38 strains sequenced, blue sites display 80 to 99% identity, and green sites
demonstrate 60 to 80% sequence identity. A white background represents amino acids not displaying
similarity with their wild-type counterpart. Sites labeled with a star are novel with respect to mutations.
The corresponding MICs of ciprofloxacin are indicated at the left.
Table 1. Antibiotic inactivation screen of the soil library. Cultures of liquid SIM supplemented with
antibiotic (20 mg/ml) were grown from spore suspensions. Supernatants were used as samples in
disk diffusion assays, and putative inactivating strains were identified by the absence of a zone of
Number of strains Complete inactivation: %
Resistant Screened for inactivationOf isolates screened Of library
*Not applicable. Statistic cannot be determined, because all resistant isolates were not assayed.
Fig. 3. Modification of
telithromycin by strain
Ja#7. (A) Culture media
mycin were analyzed by
chromatography with in-
line electrospray mass
modification of telithro-
mycin was accompanied
an increase in mass-to-
charge (m/z) ratio, and
the loss of antimicrobial
activity against the indi-
cator organism Micrococ-
cus luteus. (B) Structures of telithromycin and the Ja#7 inactivation product.
20 JANUARY 2006VOL 311SCIENCEwww.sciencemag.org
on October 1, 2007
previous studies that found natural sequence
variation within this domain in soil bacteria
(19). Knowledge of such natural variations
could complement studies on clinical isolates
to guide the rational development of next-
generation fluoroquinolones that will be active
against resistant strains.
Resistance to macrolide antibiotics in patho-
gens of clinical significance has increased
considerably over recent decades and is com-
monly a result of antibiotic efflux and ribosomal
protection mechanisms (20, 21). Substantial lev-
els of macrolide resistance were also detected
in our soil isolate library, both to the natural
product erythromycin (introduced in 1952, 27%),
and the semisynthetic telithromycin (FDA-
approved in 2004, 17%). The high frequency
of telithromycin resistance was particularly in-
triguing, because telithromycin is known for its
activity against macrolide-resistant bacteria.
Five percent of library isolates detoxified
telithromycin in culture media (Table 1). One
of these, Streptomyces strain Ja#7 (MIC of
32 mg/ml), completely modified telithromycin to
an inactive hydrophilic product with a mass of
973.6 daltons (Fig. 3). This addition of 162 dal-
tons to telithromycin (811.7 daltons) is a signa-
ture indicator of monoglycosylation. Large-scale
purification of the product, followed by multi-
analysis, confirmed that the inactive product was
2¶-O-glucosyl-telithromycin (table S1).
Modification of the cladinose 2¶-OH of
erythromycin is known to result in antibiotic
resistance (22, 23). However, Ja#7, despite its
ability to inactivate telithromycin, was unable
to completely inactivate erythromycin or its
derivative clarithromycin under identical con-
ditions. Thus, a distinct mechanism seems to be
operating. Given the abundance of resistance
determinants in streptomycetes that are homol-
ogous to those in clinically significant pathogens
(5, 24, 25), it is evident that once this mechanism
is fully characterized, it should be monitored as
telithromycin use increases clinically and resist-
ant organisms inevitably emerge.
This study provides an analysis of the
antibiotic resistance potential of soil micro-
organisms. The frequency of high-level resist-
ance seen in the study to antibiotics that have
for decades served as gold-standard treatments,
as well as those only recently approved for
human use, is remarkable. No class of antibiotic
was spared with respect to bacterial target or
natural or synthetic origin. Although this study
does not provide evidence for the direct transfer
of resistance elements from the soil resistome
to pathogenic bacteria, it identifies a previously
underappreciated density and concentration of
environmental antibiotic resistance. The level
and diversity of resistance uncovered in this
work is only partially reflective of the true
extent of the environmental resistome, because
this study was restricted exclusively to cultur-
able spore-forming bacteria, which represent
only a fraction of soil-dwelling bacteria. For
example, a recent soil metagenome analysis
uncovered several aminoglycoside resistance
genes in uncultured organisms (26). Further-
more, the primary screen was conducted at high
antibiotic concentrations, thereby excluding
phenotypes exhibiting low to intermediate re-
sistance. The level of resistance genes in the
environment is therefore very likely to be sub-
stantially higher and the antibiotic resistome
much more extensive than this study reveals.
The survey of antibiotic resistance mecha-
nisms can assist the elucidation of novel mecha-
nisms that may emerge clinically, as well as
serve as a foundation for new antibiotic devel-
opment. In addition, the study of enzymatic
inactivation could lead to the development of
inhibitors for combination therapies to restore
References and Notes
1. T. Kieser, M. J. Bibb, M. J. Buttner, K. F. Chater, D. A.
Hopwood, Practical Streptomyces Genetics (John Innes
Foundation, Norwich, UK, ed. 1, 2000).
2. B. K. Hubbard, C. T. Walsh, Angew. Chem. Int. Ed. Engl.
42, 730 (2003).
3. E. Cundliffe et al., Antonie Leeuwenhoek 79, 229 (2001).
4. J. Davies, Science 264, 375 (1994).
5. C. G. Marshall, I. A. Lessard, I. Park, G. D. Wright,
Antimicrob. Agents Chemother. 42, 2215 (1998).
6. H. J. Hong, M. S. Paget, M. J. Buttner, Mol. Microbiol. 44,
7. L. Guardabassi, A. Dalsgaard, Appl. Environ. Microbiol.
70, 984 (2004).
8. C. T. Walsh, Antibiotics: Actions, Origins, Resistance
(American Society for Microbiology Press, Washington,
DC, ed. 1, 2003).
9. H. Myllykallio, D. Leduc, J. Filee, U. Liebl, Trends
Microbiol. 11, 220 (2003).
10. D. R. Snydman, N. V. Jacobus, L. A. McDermott, J. R.
Lonks, J. M. Boyce, Antimicrob. Agents Chemother. 44,
11. I. A. Critchley et al., Antimicrob. Agents Chemother. 47,
12. W. E. Alborn Jr., N. E. Allen, D. A. Preston, Antimicrob.
Agents Chemother. 35, 2282 (1991).
13. D. Jung, A. Rozek, M. Okon, R. E. Hancock, Chem. Biol.
11, 949 (2004).
14. M. Debono et al., J. Antibiot. (Tokyo) 41, 1093 (1988).
15. M. Gellert, K. Mizuuchi, M. H. O’Dea,T. Itoh, J. I.Tomizawa,
Proc. Natl. Acad. Sci. U.S.A. 74, 4772 (1977).
16. H. Peng, K. J. Marians, J. Biol. Chem. 268, 24481 (1993).
17. L. J. Piddock, Drugs 58 (suppl. 2), 11 (1999).
18. J. H. Tran, G. A. Jacoby, Proc. Natl. Acad. Sci. U.S.A. 99,
19. B. Waters, J. Davies, Antimicrob. Agents Chemother. 41,
20. G. V. Doern et al., Antimicrob. Agents Chemother. 45,
21. P. Descheemaeker et al., J. Antimicrob. Chemother. 45,
22. N. Noguchi et al., Antimicrob. Agents Chemother. 39,
23. E. Cundliffe, Antimicrob. Agents Chemother. 36, 348 (1992).
24. C. J. Thompson, G. S. Gray, Proc. Natl. Acad. Sci. U.S.A.
80, 5190 (1983).
25. I. Chopra, M. Roberts, Microbiol. Mol. Biol. Rev. 65, 232
26. C. S. Riesenfeld, R. M. Goodman, J. Handelsman, Environ.
Microbiol. 6, 981 (2004).
27. We thank K. P. Koteva for helpful discussions and
analytical expertise; B. Ghadaki, C. Capone, and T. Patel
for help with construction of the soil library; and
S. Projan, P. Bradford, and J. Silverman for careful
reading of the manuscript. This work was funded by the
Canadian Institutes of Health Research and by a Canada
Research Chair to G.D.W. Accession numbers for
nucleotide sequences deposited in GenBank run
consecutively from DQ311010 to DQ311051.
Supporting Online Material
Materials and Methods
Figs. S1 to S4
Tables S1 to S5
30 September 2005; accepted 12 December 2005
Vaccinia Virus–Induced Cell Motility
Requires F11L-Mediated Inhibition
of RhoA Signaling
Ferran Valderrama, Joa ˜o V. Cordeiro, Sibylle Schleich, Friedrich Frischknecht,* Michael Way†
RhoA signaling plays a critical role in many cellular processes, including cell migration. Here we
show that the vaccinia F11L protein interacts directly with RhoA, inhibiting its signaling by blocking
the interaction with its downstream effectors Rho-associated kinase (ROCK) and mDia. RNA
interference–mediated depletion of F11L during infection resulted in an absence of vaccinia-
induced cell motility and inhibition of viral morphogenesis. Disruption of the RhoA binding site
in F11L, which resembles that of ROCK, led to an identical phenotype. Thus, inhibition of RhoA
signaling is required for both vaccinia morphogenesis and virus-induced cell motility.
lifetime of multicellular organisms (1, 2). De-
regulation of these two fundamental cellular
processes frequently occurs during pathological
he spatial and temporal regulation of
cell adhesion and motility is essential
during development and throughout the
situations such as tumor cell metastasis (3, 4).
Dramatic changes in cell migration and adhe-
sion, as well as loss of contact inhibition, are
also observed during many viral infections, in-
cluding that of vaccinia virus (5, 6). In contrast
to the wild-type Western-Reserve (WR) virus,
www.sciencemag.orgSCIENCEVOL 311 20 JANUARY 2006
on October 1, 2007