AbstractBackground: 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

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 ChatIsolate Cell

MorphologyGenus 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)

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

a Massol-Deya, et al. 1995 In, Methods in Molecular Microbial Ecology

What Mechanism Allows for Growth in the Presence of Lead? 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 IAre 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(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. 5 mM Pb 0 mM Pb

5 mM Pb 0 mM Pb

5 mM Pb0 mM Pb

Expectations

•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

Variants (mutants)

There is Little Evidence for Variation among Populations 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

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

0 5

Colony from high [Pb]

Colony from low [Pb]

Rhodococcus Pb5 Tolerance

0 2.5 0 5

Arthrobacter CPC2 Growth Changes over Time 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.

0 2.5

10 Days

120 Days

Variants?

The Role of Pb9(PO4)6 Precipitation

Questions Generated II

Is precipitation of Pb9(PO4)6 necessary for tolerance or resistance?

And is it truly a mutant phenotype?

Roane

Roane1 mM Pb

Streptomyces CPA1 is pink in absence of lead and brown in its presence

Streptomyces CPA1

0 2.5

Pb9(PO4)6 Precipitation is Widespread in Populations

0 5

Enterobacter aerogenes Klebsiella pneumoniae

0 5

Hyper-precipitation of Pb9(PO4)6 is a Mutant Phenotype

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

Questions Generated III Is there a correlation between heavy

metal resistance and antibiotic resistance?

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 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

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 Various Antibiotics 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.

Antibiotics used -lactam: Ampicillin,

Carbenecillin, Cefazolin, Cephatoxime, Cefaclor

Non- -lactam: Erythromycin, Kanamycin, Polymyxin B, Streptomycin, Tetracycline

Study of antibiotic resistance of chat pile isolates was done with the Kirby-Bauer Agar diffusion test

Frequency of Resistance among Isolates by Antibiotic Group

Organisms/Ab Susceptible Intermediate Resistant Total

Known/ β-lactam 8 4 38 50Known/ Non-β-

lactam 32 11 7 50Cattle Farm/β-

lactam 6 1 73 80Cattle Farm/ Non-

β-lactam 37 8 35 80Chat Pile/ β-

lactam 21 4 25 50Chat Pile/Non-β-

lactam 36 4 10 50Total 137 32 191 360

2 Contingency Table Reveals Pb Tolerant Organisms are no More Resistant than Other

Bacterial Strains

a significantly more resistant than expected, α=0.005, df=2, Fcrit= 10.6

b significantly less resistantless resistant than expected, α=0.005, df=2, Fcrit= 10.6

C significant variation among groups; α=0.005, df=10, Fcrit= 25.2

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 48.9Cattle Farm/ Non-β-lactam 1.1 0.1 1.1 2.3

Chat Pile/ β-lactam 0.1 0.0 0.0 0.2Chat Pile/Non-β-lactam 14.1 0.0 9.9 24.1

Total 50.4 15.2 53.8 119.4c

Acknowledgements

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.