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 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 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. IsolateCell Morphology Genus and species CPA1Gram+ rodsStreptomyyces (16S) (no match in FAME) CPA2Irregular Gram+ rod Arthrobacter oxydans (FAME) and (16S) CPA6Gram+Rhodococcus luteus (FAME) CPC2Irregular Gram+ rod Arthrobacter oxydans (FAME) A. globiformis (16S) CPC3Gram+ rodsStreptomyyces (16S) CPC4Irregular Gram+ rod No data – morphologically similar to Arthrobacter species CPC5Gram+No data – morphologically similar to Rhodococcus species Pb1Gram - rodPseudomonas (16S) stutzeri (Vitek) putida (FAME) Pb4Gram + rodOchrobactrum (16S) Pb5Gram+Rhodococcus fascians (16S) 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? 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) 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 II Is precipitation of Pb9(PO4)6 necessary for tolerance or resistance? And is it truly a mutant phenotype? Klebsiella pneumoniae 0 5 RESULTS Streptomyces CPA1 displays phenotype Pb 9 (PO 4 ) 6 Precipitation is Widespread in Populations Hyper-precipitation of Pb 9 (PO 4 ) 6 is a Mutant Phenotype Questions Generated III Is there a correlation between heavy metal resistance and antibiotic resistance? 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  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 less resistant 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 Organism/AbSusceptibleIntermediateResistantTotal Known/ β-lactam a Known/ Non-β-lactam b Cattle Farm/β-lactam Cattle Farm/ Non-β-lactam Chat Pile/ β-lactam Chat Pile/Non-β-lactam Total c