Antibiotic Resistance in Coastal Vibrio and E. coli - The Oceans and ...

oceansandhumanhealth.noaa.gov

Antibiotic Resistance in Coastal Vibrio and E. coli - The Oceans and ...

The role of metal contamination in the proliferation of antibiotic

resistance in coastal water-borne pathogens

OHHI - external grants

10/1/04 - 9/30/07

R Stepanauskas (lead PI). Bigelow Laboratory for Ocean Sciences.

JV McArthur; C Baker-Austin, TC Glenn; CH Jagoe, A Lindell. Savannah River Ecology Laboratory

ME Frischer. Skidaway Institute of Oceanography.

JA Gooch. NOAA/ CCEHBR.

CR Tuckfield. Savannah River National Laboratory

J Ravel and W Fricke, TIGR


Molecular mechanisms of metal and

antibiotic resistance co-selection

Baker-Austin et al. 2006


Molecular mechanisms of metal and

antibiotic resistance co-selection

Cross-resistance

Baker-Austin et al. 2006


Molecular mechanisms of metal and

antibiotic resistance co-selection

Cross-resistance

Co-resistance

Baker-Austin et al. 2006


Molecular mechanisms of metal and

antibiotic resistance co-selection

Cross-resistance

Co-resistance

Baker-Austin et al. 2006

Co-expression


Study approach:

•Field monitoring

•Microcosm experiments

•Genome analyses


Studied tidal systems

Ship Yard Creek, Charleston, SC.

• Former nuclear submarine site

• Extensively polluted with a mixture of heavy

metals, including chromium, mercury, and

cadmium.

• Designated special EPA ‘Superfund status’

due to pollution

NC

SC

GA

FL

The ACE Basin, Beaufort, SC.

• Reference site

• No history of industrial or urban

contamination.

• Considered a pristine estuary - designated

a national wildlife refuge.

LCP Site, Brunswick, GA

• Former industrial complex - made fish

poison, bleach and chloro-alkali compounds,

now disused.

• Extensively mercury polluted site -

considered one of the worst in USA.

• Designated special EPA ‘Superfund status’

due to pollution


Studied tidal systems

Ship Yard Creek, Charleston, SC.

• Former nuclear submarine site

• Extensively polluted with a mixture of heavy

metals, including chromium, mercury, and

cadmium.

• Designated special EPA ‘Superfund status’

due to pollution

NC

SC

GA

FL

The ACE Basin, Beaufort, SC.

• Reference site

• No history of industrial or urban

contamination.

• Considered a pristine estuary - designated

a national wildlife refuge.

Antibiotic resistance in

E.coli, V. vulnificus, and V.

parahaemolyticus

LCP Site, Brunswick, GA

• Former industrial complex - made fish

poison, bleach and chloro-alkali compounds,

now disused.

• Extensively mercury polluted site -

considered one of the worst in USA.

• Designated special EPA ‘Superfund status’

due to pollution


Studied tidal systems

Ship Yard Creek, Charleston, SC.

• Former nuclear submarine site

• Extensively polluted with a mixture of heavy

metals, including chromium, mercury, and

cadmium.

• Designated special EPA ‘Superfund status’

due to pollution

NC

SC

GA

FL

The ACE Basin, Beaufort, SC.

• Reference site

• No history of industrial or urban

contamination.

• Considered a pristine estuary - designated

a national wildlife refuge.

Antibiotic resistance in

E.coli, V. vulnificus, and V.

parahaemolyticus

• Integrases in the

metagenome

LCP Site, Brunswick, GA

• Former industrial complex - made fish

poison, bleach and chloro-alkali compounds,

now disused.

• Extensively mercury polluted site -

considered one of the worst in USA.

• Designated special EPA ‘Superfund status’

due to pollution


Studied tidal systems

Ship Yard Creek, Charleston, SC.

• Former nuclear submarine site

• Extensively polluted with a mixture of heavy

metals, including chromium, mercury, and

cadmium.

• Designated special EPA ‘Superfund status’

due to pollution

NC

SC

GA

FL

The ACE Basin, Beaufort, SC.

• Reference site

• No history of industrial or urban

contamination.

• Considered a pristine estuary - designated

a national wildlife refuge.

Antibiotic resistance in

E.coli, V. vulnificus, and V.

parahaemolyticus

• Integrases in the

metagenome

• Various seasons

LCP Site, Brunswick, GA

• Former industrial complex - made fish

poison, bleach and chloro-alkali compounds,

now disused.

• Extensively mercury polluted site -

considered one of the worst in USA.

• Designated special EPA ‘Superfund status’

due to pollution


Studied tidal systems

Ship Yard Creek, Charleston, SC.

• Former nuclear submarine site

• Extensively polluted with a mixture of heavy

metals, including chromium, mercury, and

cadmium.

• Designated special EPA ‘Superfund status’

due to pollution

NC

SC

GA

FL

The ACE Basin, Beaufort, SC.

• Reference site

• No history of industrial or urban

contamination.

• Considered a pristine estuary - designated

a national wildlife refuge.

Antibiotic resistance in

E.coli, V. vulnificus, and V.

parahaemolyticus

• Integrases in the

metagenome

• Various seasons

• Sediment, ebb and flood

samples

LCP Site, Brunswick, GA

• Former industrial complex - made fish

poison, bleach and chloro-alkali compounds,

now disused.

• Extensively mercury polluted site -

considered one of the worst in USA.

• Designated special EPA ‘Superfund status’

due to pollution


Tidal creek sample collection

Tides


Tidal creek sample collection

Sediment

samples

Tides


Tidal creek sample collection

Sediment

samples

Tides

Flood and ebb

water samples


Antibiotic resistance in E. coli

Percent of Phenotypes

Ak

Apr

Gm

St

Imp

Mer

Cex

Cax

Cf

Cfx

T

T/S

E

Azi

Fd

Am

Amx

P

C

Cp

Mox

NA

Ofl

Sz

Otet

Te

100

80

60

40

20

0

P

E

Amikacin

Apramycin

Gentamicin

Streptomycin

Imipenem

Meropenem

Cephalexin

Ceftriaxone

Cephalothin

Cefoxitin

Trimethoprim

Trimethoprim/Sulfa

Erythromycin

Azithromycin

Nitrofurantoin

Ampicillin

Amoxicillin

Penicillin

Chloramphenicol

Ciprofloxacin

Moxifloxacin

Nalidixic Acid

Ofloxacin

Sulfathiazole

Oxytetracycline

Tetracycline

T

Otet

St

Te

Sz

Cfx

Fd

Cex

Apr

Am

Cf

Amx

Azi

Resistances to 22 antibiotics

245 strains analyzed

Shipyard Creek

superfund site

Antibiotics

Percent of Phenotypes

C

Ak

Mox

Gm Imp

Ofl

Cp

NA

100

80

60

40

20

0

P

E

T

Otet

St

Te

Sz

Cfx

Fd

Cex

Apr

Am

Cf

Amx

Azi

100

80

60

40

20

0

P

E

T

Otet

St

Te

Sz

Cfx

Fd

Cex

Apr

Am

Cf

Amx

Azi

Resistances to 14 antibiotics

124 strains analyzed

Antibiotics

ACE basin

(reference site)

C

Ak

Mox

Gm Imp

Ofl

Cp

NA

Resistances to 18 antibiotics

63 strains analyzed

LCP Chemical

superfund site

Antibiotics

C

Ak

Mox

Gm Imp

Ofl

Cp

NA


E. coli resistance diversity

ACE basin

reference site

LCP chemical

superfund site

Shipyard Creek

superfund site

Figure legend

Ak

Amikacin

Azi

Azithromycin

Apr

Apramycin

Fd

Nitrofurantoin

Gm

Gentamicin

Am

Ampicillin

St

Streptomycin

Amx

Amoxicillin

Imp

Imipenem

P

Penicillin

Mer

Meropenem

C

Chloramphenicol

Cex

Cephalexin

Cp

Ciprofloxacin

Cax

Ceftriaxone

Mox

Moxifloxacin

Cf

Cephalothin

NA

Nalidixic Acid

Cfx

Cefoxitin

Ofl

Ofloxacin

T

Trimethoprim

Sz

Sulfathiazole

T/S

Trimethoprim/Sulfa

Otet

Oxytetracycline

E

Erythromycin

Te

Tetracycline


Class 1 integron abundance, qPCR data

10 -1 ACE

Contaminated sites

LCP

S YC

10 -2

IntI1:16S rRNA genes

10 -3

10 -4

10 -5

ebb flood sed


Evidence for indirect selection

in estuaries


Evidence for indirect selection

in estuaries

For:


Evidence for indirect selection

in estuaries

For:

• More antibiotic resistance (AR) in E. coli in contaminated creeks


Evidence for indirect selection

in estuaries

For:

• More antibiotic resistance (AR) in E. coli in contaminated creeks

• More integrons in contaminated creeks


Evidence for indirect selection

in estuaries

For:

• More antibiotic resistance (AR) in E. coli in contaminated creeks

• More integrons in contaminated creeks

Against:


Evidence for indirect selection

in estuaries

For:

• More antibiotic resistance (AR) in E. coli in contaminated creeks

• More integrons in contaminated creeks

Against:

• AR in V. vulnifcus and V. parahaemolyticus is not higher in

contaminated sites relative to the reference site


Evidence for indirect selection

in estuaries

For:

• More antibiotic resistance (AR) in E. coli in contaminated creeks

• More integrons in contaminated creeks

Against:

• AR in V. vulnifcus and V. parahaemolyticus is not higher in

contaminated sites relative to the reference site

• Isolate AR and integron abundance is not higher in sediment and

ebb samples compared to flood samples


Conclusions


Conclusions

• No evidence for indirect selection in the studied tidal systems


Conclusions

• No evidence for indirect selection in the studied tidal systems

• Plausible explanation for the higher AR in E. coli and higher abundance

of integrons in the contaminated sites:


Conclusions

• No evidence for indirect selection in the studied tidal systems

• Plausible explanation for the higher AR in E. coli and higher abundance

of integrons in the contaminated sites:

• Movement of multi-resistant microorganisms from urban areas

adjacent to the studied metal-contaminated sites:


Conclusions

• No evidence for indirect selection in the studied tidal systems

• Plausible explanation for the higher AR in E. coli and higher abundance

of integrons in the contaminated sites:

• Movement of multi-resistant microorganisms from urban areas

adjacent to the studied metal-contaminated sites:

• In support, human polyoma virus was detected in Shipyard

Creek samples, indicative of sewage contamination,


Conclusions

• No evidence for indirect selection in the studied tidal systems

• Plausible explanation for the higher AR in E. coli and higher abundance

of integrons in the contaminated sites:

• Movement of multi-resistant microorganisms from urban areas

adjacent to the studied metal-contaminated sites:

• In support, human polyoma virus was detected in Shipyard

Creek samples, indicative of sewage contamination,


Plausible explanation for undetected metal effects in estuaries:

Low metal bioavailability in seawater

Cd 2+ speciation (%) in:

Freshwater, pH 6.0 Seawater, pH 8.2


Plausible explanation for undetected metal effects in estuaries:

Low metal bioavailability in seawater

Cd 2+ speciation (%) in:

Freshwater, pH 6.0 Seawater, pH 8.2

100

75

50

25

0

Free OH Cl SO4 CO3


Plausible explanation for undetected metal effects in estuaries:

Low metal bioavailability in seawater

Cd 2+ speciation (%) in:

Freshwater, pH 6.0 Seawater, pH 8.2

100

75

50

25

0

Free OH Cl SO4 CO3


Plausible explanation for undetected metal effects in estuaries:

Low metal bioavailability in seawater

Cd 2+ speciation (%) in:

Freshwater, pH 6.0 Seawater, pH 8.2

100

75

50

25

0

Free OH Cl SO4 CO3


Plausible explanation for undetected metal effects in estuaries:

Low metal bioavailability in seawater

Cd 2+ speciation (%) in:

Freshwater, pH 6.0 Seawater, pH 8.2

100

75

50

25

0

Free OH Cl SO4 CO3


Microcosm experiments


Microcosm results:

Frequency of multi-resistant isolates

Seawater microcosms

Frequency of multiple resistances, % of isolates

100

75

50

25

0

Hg

Cd

0 0.1 1 10 100

Metal concentration in microcosms, M



Cd


Cr


Microcosm results:

Frequency of multi-resistant isolates

Frequency of multiple resistances, % of isolates

100

75

50

25

0

Seawater microcosms

Hg

Cd

0 0.1 1 10 100

Metal concentration in microcosms, M



Cd


Cr

Freshwater microcosms

*

Cd

*

Ni

*

Tetracycline

Ampicillin


Microcosm results:

Frequency of multi-resistant isolates

Frequency of multiple resistances, % of isolates

100

75

50

25

0

Seawater microcosms

Hg

Cd

0 0.1 1 10 100

Metal concentration in microcosms, M



Cd


Cr

Freshwater microcosms

*

Cd

*

Ni

*

Tetracycline

Ampicillin


Antibiotic resistance in V. vulnificus

(150 isolates)


Antibiotic resistance in V. vulnificus

(150 isolates)


Antibiotic resistance in V. vulnificus

(150 isolates)


Genomics of a multi-resistant

E. coli strain SMS-3-5

• Resistant to 33 out of 43 tested antibiotics

• First environmental case of resistance to frontline fluoroquinolones:

ciprofloxacin, levofloxacin, norfloxacin, sparfloxacin, and

moxifloxacin

Chromosome Largest plasmid Genes involved in

high-level

fluoroquinolone

resistance:

fsr

Acr/Bcr

ompF

MFS

pmrD

smr


Take-home messages


Take-home messages

No evidence of indirect selection in estuaries


Take-home messages

No evidence of indirect selection in estuaries

High antibiotic resistance in coastal pathogens


Take-home messages

No evidence of indirect selection in estuaries

High antibiotic resistance in coastal pathogens

Novel mechanisms of resistance to fluoroquinolones

More magazines by this user
Similar magazines