Transcript Abstract - Southeast Missouri State University

Abstract
Background: Bacteria able to grow in the presence of Pb are of interest for bioremediation and
insight into mechanisms of defense against the heavy metal. Three aspects were studied: 1) how
widespread in a population is growth at elevated [Pb]; 2) how universal is the deposition of
Pb9(PbO4)6 resulting in brown colony morphology (O’ Brien WF et al. Abstr. 103rd Gen. Meet.
ASM, abstr. O-129, 2003.); and 3) how does growth correlate to antibiotic resistance? Methods:
The first two questions were investigated in 10 bacteria isolated from chat by spreading TSA-grown
cell suspensions on defined minimal media (Roane media, RM) containing gradient of 0-2.5 mM
Pb. Resistance to 17 antibiotics was assayed by the Kirby-Bauer Agar method on Mueller-Hinton
plates. The identity of environmental isolates was investigated by Blast sequence analysis of PCR
amplified 16S rDNA. Results and Discussion: Three independent isolates of Rhodococcus
fascians showed no decrease in viability across gradient plates and no change in colony
morphology. This suggests that this species is lead tolerant and the ability to grow in the presence
of lead is a widespread physiological trait of the organism. On the other hand strains of
Pseudomonas (veronii or reactans by 16S sequence), Ochrobactrum, 2 isolates of Arthrobacter
(either oxydans or polychromogenes by 16S sequence), and independent isolates of a novel
organism (CPA1and CPC3) all showed decrease in viability across the gradient. The ability to
grow at elevated [Pb] was limited to select individuals in the population which would indicate a
genetic basis for resistance. CPA1 and CPC3 coloration was seen to change from pink to brown at
the higher concentrations of Lead. Also, we have observed this color shift in general populations of
Enterobacter. This phenotype was first reported in mutants of V. harveyi and Caulobacter
crecentus, and we have isolated a Klebsiella pneumoniae mutant. Antibiotic test results have been
collected from the 10 Pb-mine isolates, 10 known cultures, and 17 Ampicillin resistant bacteria
from a cattle-farm, and Principal Component Analysis will be used to assess patterns of resistance
in the 3 groups.
Lead Mining in Missouri
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East central Missouri is known as
the lead-belt region. Chat is the
byproduct of mining lead ore.
The lead concentration was
measured at 16,00 ppm; 4,000
ppm in the surrounding soil.
Hypothesis: Chat represents a
natural enrichment for bacteria
that can withstand the toxic
effects of Pb.
10 bacterial Isolates from this
environment are characterized as
to their response to Pb in this
presentation
Identification of Microorganisms Recovered from Chat
Isolate
Cell
Morphology
Genus and species
CPA1
Gram+ rods
Streptomyyces (16S)
(no match in FAME)
CPA2
Irregular Gram+
rod
Arthrobacter oxydans (FAME)
and (16S)
CPA6
Gram+
Rhodococcus luteus (FAME)
CPC2
Irregular Gram+
rod
Arthrobacter oxydans (FAME)
A. globiformis (16S)
CPC3
Gram+ rods
Streptomyyces (16S)
CPC4
Irregular Gram+
rod
No data – morphologically
similar to Arthrobacter species
CPC5
Gram+
No data – morphologically
similar to Rhodococcus species
Pb1
Gram - rod
Pseudomonas (16S) stutzeri
(Vitek) putida (FAME)
Pb4
Gram + rod
Ochrobactrum (16S)
Pb5
Gram+
Rhodococcus fascians (16S)
a
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Identities determined by 16S
rDNA sequence, or FAME, or
both
16S rDNA genes were
amplified with primers A and
Ha. Amplicons were ligated
into pCR2.1, and inserts were
sequence using M13 forward
and reverse primers
(BioResource Center, Cornell
University).
Sequences for isolates were
compared with those in
GenBank using the Blast
protocol
Massol-Deya, et al. 1995 In, Methods in Molecular Microbial Ecology
What Mechanism Allows for Growth
in the Presence of Lead?
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Many strains of bacteria
grow in the presence of
lead.
Ralstonia metallidurans
has a plasmid encoded
metal resitance ATPase. No
plasmid have been detected
in these isolates.
Caulobacter and Vibrio
mutants show precipitation
of lead as Pb9(PO4)6
(O’Brien et al., 2003)
Questions Generated I
Are populations uniform with
regards to growth in the
presence of lead?
Are clones isolated at higher
lead concentrations “true
breeding” with regards to
tolerance/resistance?
Approach - Gradient Plates
5 mM Pb
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5 mM Pb(II)NO3 Media
(represented by red agar in
illustration) is poured with
the plates on a slant, and are
cooled.
The plates are laid flat and
and equal volume of Media
with no Pb (represented by
blue agar in illustration) is
poured atop.
0 mM Pb
0 mM Pb
5 mM Pb
5 mM Pb
0 mM Pb
Expectations
Variants (mutants)
•Non-specific defense, Structural or
Physiological Property of Organism
•Tolerance, population uniform in
response to lead
•Specific defense mechanism
•Resistance, population polymorphic
with regards to response to lead
There is Little Evidence for Variation
among Populations
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Pseudomonas Pb1 shown here does show
colonies past the general zone of growth
suggesting the population is generally
tolerant.
Upon reflection, the statement in our
abstract “On the other hand strains of
Pseudomonas (veronii or reactans by 16S
sequence), Ochrobactrum, 2 isolates of
Arthrobacter (either oxydans or
polychromogenes by 16S sequence), and
independent isolates of a novel organism
(CPA1and CPC3) all showed decrease in
viability across the gradient.” is in error.
We suggest populations are uniform in
their response to Pb and are tolerant, rather
resistant.
0
5
Ochrobactrum Pb4 Colonies that
Survive High [Pb] are Not More Fit
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Colony from
high [Pb]
Colony from
low [Pb]
0
5
To test if clones are “true
breeding”, colonies were picked
from areas of high lead
concentrations and plated side by
side with those from areas of low
concentration on new gradient
plates.
Colonies from either environment
did equally well
Rhodococcus Pb5 Tolerance
0
2.5
0
5
Arthrobacter CPC2 Growth Changes
over Time
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Arthrobacter strains often would
show additional colonies after
extended incubation
Although initially interpreted as
resistant mutants, these may
simply be survivors that
maintained viability until the Pb
concentration dissipated by
diffusion or interaction with other
colonies.
They may still be resistant
mutants however.
10 Days
120 Days
Variants?
0
2.5
The Role of Pb9(PO4)6 Precipitation
Roane
Roane
1 mM Pb
Streptomyces CPA1 is pink in absence
of lead and brown in its presence
Questions Generated II
Is precipitation of
Pb9(PO4)6 necessary for
tolerance or resistance?
And is it truly a mutant
phenotype?
Streptomyces CPA1
0
2.5
Pb9(PO4)6 Precipitation is Widespread in
Populations
Enterobacter aerogenes
0
Klebsiella pneumoniae
5
0
5
Hyper-precipitation of Pb9(PO4)6 is a
Mutant Phenotype
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K. pneumoniae
mutants were
observed that
expressed much
more pigmentation
Wild type colonies
are colorless on this
plate
Deeply pigmented variants
Antibiotics Resistance in Chat Isolates
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The observation that there is a
correlation between metal
“resistance” and antibiotic resistance
has been reported by several authors
(e.g Francis et al. Abstr. 103rd Gen.
Meet. ASM, abstr.Q-416, 2003)
Possible mechanisms may include
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Common cell feature, e.g. EPS
excludes both toxic metal and
antibiotic
 More specific feature such as an
efflux pump may exclude both
materials
 Exposure to mixed waste containing
both metals and antibiotics from the
environment selects for both traits
Questions Generated III
Is there a correlation between heavy
metal resistance and antibiotic
resistance?
Note: On the advice of more sober
statisticians we have used Chi-square
contingency tables rather than Principal
Component Analysis to analyze the data
as mentioned in our abstract.
Comparison of Frequency of Resistance to
Study of antibiotic resistance of chat
Various Antibiotics
pile isolates was done with the KirbyBauer Agar diffusion test
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Organisms used
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Chat Pile Lead-mine tailings
isolates (focus of this study): 10
organisms including Rhodococcus,
Pseudomonas, Streptomyces,
Ochrobactrum, and Arthrobacter
 Reference organisms associated
with soil (Lab teaching strains): B.
cereus, B. megaterium, B. subtilis,
B. brevis, B. pumilis, P.
aeruginosa, P. putida, P.
fluorescens, P. paucimobilis, P.
stutzeri
 Cattle farm ampicillin resistant
isolates (see Q-184): 16 organisms
including Chryseobacterium,
Pseudomonas, Aeromonas,
Morganella, and Escherichia.
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Antibiotics used
-lactam: Ampicillin,
Carbenecillin, Cefazolin,
Cephatoxime, Cefaclor
 Non- -lactam: Erythromycin,
Kanamycin, Polymyxin B,
Streptomycin, Tetracycline
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Frequency of Resistance among Isolates by
Antibiotic Group
Organisms/Ab
Susceptible
Intermediate
Resistant
Total
Known/ β-lactam
8
32
6
37
21
36
137
4
11
1
8
4
4
32
38
7
73
35
25
10
191
50
50
80
80
50
50
360
Known/ Non-βlactam
Cattle Farm/βlactam
Cattle Farm/
Non-β-lactam
Chat Pile/ βlactam
Chat Pile/Non-βlactam
Total
2 Contingency Table Reveals Pb Tolerant
Organisms are no More Resistant than Other
Bacterial Strains
Organism/Ab
Susceptible
Intermediate
Resistant
Total
Known/ β-lactam
6.7
0.0
5.4
12.2a
Known/ Non-β-lactam
8.1
9.7
14.0
31.8b
Cattle Farm/β-lactam
20.3
5.3
23.3
48.9
Cattle Farm/ Non-β-lactam
1.1
0.1
1.1
2.3
Chat Pile/ β-lactam
0.1
0.0
0.0
0.2
Chat Pile/Non-β-lactam
14.1
0.0
9.9
24.1
Total
50.4
15.2
53.8
119.4c
a
significantly more resistant than expected, α=0.005, df=2, Fcrit= 10.6
b
significantly less resistant than expected, α=0.005, df=2, Fcrit= 10.6
C
significant variation among groups; α=0.005, df=10, Fcrit= 25.2
Acknowledgements
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We would like to thank our co-authors not in attendance Saira Khan (not pictured), Andrew
Corcoran (above left), Jennifer Seabaugh (at base of chat pile), and Mary Hobbs (above right).
Funding for Joe Toney’s travel was made available through the Southeast Missouri State
University Student Professional Development program Drs. Rick Burns and Christina Frazier.
Antibiotic resistance data on cattle farm isolates and laboratory strains of bacteria was made
available by Kimberleigh Foster and Melanie Miller respectively.
The authors would like to thank the following people for their excellent technical assistance:
Maija Bluma, Mindy Hoffman, and Tim Capps. Also, Vicki Howell and Joanna Kubik
provided administrative support.