Transcript a 2-sided "mini-poster" version. - Southeast Missouri State University

Growth, Pigmentation, and Antibiotic Resistance Phenotypes of Bacteria Grown in the Presence of Lead
JOE TONEY, S. KHAN, A. CORCORAN, J. SEABAUGH, M. HOBBS, and JAMES E. CHAMPINE
Southeast Missouri State University, Cape Girardeau, MO 63701 ([email protected])
Abstract Q-201 (section in red italics now considered incorrect)
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.
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)
RESULTS
•There is Little Evidence for Variation among Populations
•Ochrobactrum Pb4 Colonies that Survive High [Pb] are
Not More Fit
•Arthrobacter CPC2 Growth Changes over Time
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?
•Specific defense mechanism
•Resistance, population
polymorphic with regards to
response to lead
Genus and species
•Non-specific defense,
Structural or Physiological
Property of Organism
•Tolerance, population
uniform in response to lead
Cell
Morphology
Variants (mutants)
Isolate
Questions Generated II
Is precipitation of Pb9(PO4)6 necessary for
tolerance or resistance?
And is it truly a mutant phenotype?
Klebsiella pneumoniae
RESULTS
•Streptomyces CPA1 displays phenotype
•Pb9(PO4)6 Precipitation is Widespread in
Populations
•Hyper-precipitation of Pb9(PO4)6 is a
Mutant Phenotype
Questions Generated III
Is there a correlation between heavy
metal resistance and antibiotic
resistance?
0
Organisms used
•
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
5
Antibiotics used
•
-lactam: Ampicillin,
Carbenecillin, Cefazolin,
Cephatoxime, Cefaclor
•
Non- -lactam: Erythromycin,
Kanamycin, Polymyxin B,
Streptomycin, Tetracycline
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