Abstract

1. 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 Rhodococcusfascians 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.

2. 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

3. Identification of Microorganisms Recovered from Chat • 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

4. Many strains of bacteria grow in the presence of lead. Ralstonia metallidurans has aplasmid 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? What Mechanism Allows for Growth in the Presence of Lead?

5. Approach - Gradient Plates 5 mM Pb 0 mM Pb • 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

6. 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

7. 0 5 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.

8. 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 Colony from high [Pb] Colony from low [Pb]

9. Rhodococcus Pb5 Tolerance 0 2.5 0 5

10. 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. 10 Days 120 Days Variants? 0 2.5

11. The Role of Pb9(PO4)6 Precipitation Roane Questions Generated II Is precipitation of Pb9(PO4)6necessary for tolerance or resistance? And is it truly a mutant phenotype? Roane 1 mM Pb Streptomyces CPA1 is pink in absence of lead and brown in its presence

12. Streptomyces CPA1 0 2.5

13. 0 0 5 5 Pb9(PO4)6 Precipitation is Widespread in Populations Enterobacter aerogenes Klebsiella pneumoniae

14. Hyper-precipitation of Pb9(PO4)6is a Mutant Phenotype • K. pneumoniae mutants were observed that expressed much more pigmentation • Wild type colonies are colorless on this plate Deeply pigmented variants

15. 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 Antibiotics Resistance in Chat Isolates 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.

16. 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 Comparison of Frequency of Resistance to Various Antibiotics Study of antibiotic resistance of chat pile isolates was done with the Kirby-Bauer Agar diffusion test

17. Frequency of Resistance among Isolates by Antibiotic Group

18. 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 resistant than expected, α=0.005, df=2, Fcrit= 10.6 C significant variation among groups; α=0.005, df=10, Fcrit= 25.2

19. 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.