Creek Sediments as a Rich Source of BioSafety Level-1 Organisms for Use as Unknowns in an Undergraduate Microbiology Laboratory. Jeffrey D. Newman. Lycoming College, Williamsport, PA.

Abstract 2004 & 2005 2006 2007 Table 1. Unknown Organisms Identified by the Spring, ‘04 & ’05 Microbiology Classes Table 2. Unknown Organisms Identified by the Spring, 2006 Microbiology Class Table 3. Unknown Organisms Identified by the Spring, 2007 Microbiology Class Most general Microbiology courses include some type of project in which isolates 16S rRNA 16S 16S 16S 16S BSL species rRNA BSL species rRNA 2004 2005 BSL species % ID rRNA rRNA students must identify an organism that is unknown to them. Often, the identity % ID % ID BSL species % ID BSL species % ID 1 - Arthrobacter nitroguaiacolicus 98 of students’ “unknowns” are known by the instructor, putting pressure on the - Corynebacterium sp. nov. 94 1 Ochrobactrum tritici 99.5 - Arthrobacter bergeri 99 1 Janthinobacterium lividum >99 3 1 Flexibacteriaceae gen nov. sp. nov. ~91 - Gordonia amicalis 98.5 1 Comamonas kersterii 98.5 - Arthrobacter koreensis >97 1 Aeromonas salmonicida 99 student to get the right answer and making the activity more like a test than 1 - Bacillus aquamarinus 97.5 1 Arthrobacter oxydans 99 1 Janthinobacterium lividum 98 - Arthrobacter nitroguaiacolicus 98.5 1 Aeromonas sobria 100 investigative science. In this study, microbes were obtained from creek 2 - Bacillus arvi 99.5-99.8 - Arthrobacter rhombi 99 - Chromobacterium sp. nov. 96 - Arthrobacter sp. bad - Aeromonas sp. bad sediments and used as unknowns in a Microbiology course to test the hypothesis 4 1 Bacillus flexus 97.5-99.3 1 Microbacterium flavescens 98 2 Aeromonas hydrophila 98.5 1 Curtobacterium citreum 98 1 Shewanella putrefaciens 99 2 1 Bacillus licheniformis 99.5-99.8 that this real research experience will engage students and increase the 1 Sanguibacter keddieii 100 1 Buttiauxella agrestis 99 Microbacterium 1 1 - Bacillus muralis 99-100 likelihood that they will continue in Microbiology. 1 Streptomyces avermitis 98.5 1 Buttiauxella izardii 99.3 1 esteraromaticum >97 1 Enterobacter asburiae >97 12 11 1 Bacillus mycoides 97.2-100 - Chryseobacterium sp. nov 94 1 Enterobacter asburiae 99 1 Oerskovia turbata 99 1 Hafnia alvei >98 1 3 - Bacillus odysseyi 98.3-99.0 1 Flavobacterium hibernum 98 1 Serratia fonticola 99 1 Flavobacterium hibernum 98 1,2 Kluyvera cryocrescens 98 Samples were obtained from a variety of sites along the Loyalsock Creek in 6 2 1 Bacillus pumilus 99.3-100 - Flavobacterium sp. nov. 95.3 1,2 Acinetobacter calcoaceticus 97.6 - Flavobacterium sp. nov. 96 1 Pantoea agglomerans 98 5 1 Bacillus subtilis 98.8-100 PA and spread on TSA plates. Isolated colonies were then purified and used as 2 Sphingobacterium faecium 98.8 - Acinetobacter sp. nov. 95 - Flavobacterium sp. nov. 95 1 Pantoea ananatis bad 1 - Bacillus sylvestris 98.8 unknown organisms in a sophomore-junior level Microbiology course. During the - Bacillus arvi 99.3 1 Pseudomonas fragi 99.1 - Sejongia sp. nov. 95 1 Pseudomonas veronii 100 1 1 Paenibacillus amylolyticus 99.5 eight weeks of unknown microbe characterization, students performed Pseudomonas 1 Bacillus flexus >97 1 Pseudomonas fulva >99 1 1,2 Staphylococcus epidermidis 100 1 Bacillus flexus 99 - 98.8 frederiksbergensis - Bacillus muralis >98 1 Pseudomonas jessenii >97 microscopic analysis, biochemical, physiological and antibiotic sensitivity tests 1 2 Staphylococcus haemolyticus 99.5 1 Bacillus licheniformis 97.5 1 Pseudomonas graminis 98.5 - Bacillus sp. nov. 96 - Pseudomonas libanensis >98 and 16S rRNA gene sequence analysis to identify their organisms. During 2004 4 10 1 Serratia fonticola 97.5-100 1 Bacillus pumilus 100 - Pseudomonas kilonensis 99 1 Bacillus pumilis 98.5 - Pseudomonas proteolytica 98 3 1 Serratia grimesii/proteomaculans 99.0-99.5 Exiguobacterium and 2005, when students themselves chose colonies from the spread plates, 1 98.8 - Pseudomonas libanensis 99 1 Bacillus subtilis >97 - Pseudomonas umsongensis 97 1 - Serratia novum 96 oxidotolerans many duplicate unknowns were obtained. Only 23 different species were - Exiguobacterium undae 97 5 1 Serratia plymuthica 99.5-100 1 Paenibacillus cookii 98 - Pseudomonas proteolytica 98.5 identified by the 91 students. Nearly 60% of students had members of the genus 1 Paenibacillus amylolyticus 98 5 2 Yersinia intermedia 97.5-99.8 1 Staphylococcus caprae 99.8 - Pseudomonas sp. bad 2 Staphylococcus pasteuri 99 Bacillus and another 25% had one of four Serratia species. In 2006 and 2007, 1 - Pseudomonas koreensis 99.5 1 Carnobacterium viridans 98.5 1 Pseudomonas syringae bad morphologically distinguishable colonies were chosen from spread plates and 3 - Pseudomonas libanensis 98.5 Bacillus flexus CVB Aeromonas salmonicida MR Bacillus flexus MAM Aeromonas sp. ELP streaked by the instructor to maximize the diversity of unknowns. Of the 89 1 - Pseudomonas mediterranea 99 Bacillus arvi TLF Aeromonas salmonicida ABL Aeromonads Exiguobacterium oxidotolerans CNH Aeromonas sobria DJBH unknown organisms identified in 2006 and 2007, 66 different species were Carnobacterium viridans AJR Aeromonas salmonicida KLK Bacillus subtilis DD Carnobacterium viridans ZRT Shewanella putrefaciens CLH obtained, from 30 genera and 20 families. Nine of these were novel species based Bacillus subtilis SEI Firmicutes Bacillus subtilis JLC Bacillus pumilis JD Shewanella putrefaciens KMF Alteromonads on having <97% sequence identity to type strains, and only 5 were known BSL-2 Bacillus subtilis CI Bacillus licheniformis JAS Enterobacter asburiae BWW Bacillus subtilis SR Bacillus pumilis TCB Kluyvera cryocrescens AMG Bacillus licheniformis BLM organisms. Bacillus licheniformis OJE Staphylococcus caprae AB Hafnia alvei TJC Enterobacteria Bacillus pumilis AS Paenibacillus cookii GMB Pantoea agglomerans AMC Bacillus pumilis LJ Arthrobacter oxydans JNT Pantoea agglomerans BMG Bacillus pumilis ENL Pseudomonas fulva NMG Student learning was assessed using research style lab reports sent to two Bacillus pumilis KAS Arthrobacter rhombi PH Bacillus pumilis EKC Microbacterium flavescens JB Pseudomonas umsongensis DAB other class members for anonymous peer-review and revised before submission Bacillus pumilis JLH Sanguibacter keddieii CTL Actinobacteria Pseudomonas jessenii REB Bacillus pumilis KC Streptomyces avermitis SMC Pseudomonas proteolytica AMW Pseudomonads Bacillus odysseyi AMS for grading. Course evaluation surveys have been very positive. Several students Bacillus odysseyi LLP Corynebacterium sp. nov PH Pseudomonas veronii SLK Bacillus odysseyi ANR Gordonia amicalis JLB Pseudomonas libaniensis KWH have chosen to follow-up on the novel organisms as part of Independent Studies, Bacillus odysseyi RBB Ochrobactrum tritici-grignonense AJF -proteobacteria Janthinobacterium lividum JLB -proteobacteria Bacillus aquamarinus AS Bacillus flexus BLM Honor’s Projects, or the Cell & Molecular Biology Research Methods course and Bacillus arvi BJS Pseudomonas graminis CAK Bacillus arvi PH Pseudomonas graminis SAR Bacillus muralis CAH Bacillus sylvestris MJR Pseudomonas fragi KKB Bacillus subtilis CMD will present their work at the 2007 ASM General Meeting. Bacillus flexus BMF Pseudomonas frederiksbergensis TDM Bacillus pumilis EJU Bacillus flexus NF Pseudomonads Firmicutes Bacillus flexus ARB Pseudomonas kilonensis SKR Bacillus sp. nov. SLS Bacillus flexus JM Firmicutes Pseudomonas proteolytica JMR Staphylococcus pasteuri VJW Bacillus muralis NP Pseudomonas libanensis MPW Exiguobacterium undae SLD Bacillus muralis EP Bacillus mycoides EMH Acinetobacter calcoaceticus TRM Paenibacillus amylolyticus LEB Bacillus mycoides MAZ Acinetobacter sp. nov MAG Moraxellaceae Arthrobacter bergeri MRK Bacillus mycoides EMW Buttiauxella agrestis KAM Arthrobacter bergeri NAW Bacillus mycoides MD Curtobacterium citreum GMT Actino- Bacillus mycoides DPC Buttiauxella izardii SEG Enterobacteria Unknown Microbe Identification Experiment Bacillus mycoides RJF Enterobacter asburiae AMC Microbacterium esteraromaticum JMB Bacillus mycoides DLK Serratia fonticola BRM Oerskovia turbata CJP bacteria Bacillus mycoides EMP Arthrobacter koreensis JWG Bacillus mycoides EW Aeromonas hydrophila JMT Aeromonads Arthrobacter nitroguaiacolicus DVG Project Objectives: Bacillus mycoides SZ Chromobacterium sp. nov JWM Bacillus mycoides EKS Janthinobacterium lividum LAR -proteobacteria Flavobacterium hibernum LES Bacillus mycoides NK Comamonas kersterii DKS Flavobacterium sp. nov. LMK • Engage students in an investigative research experience. Bacillus mycoides EK Flavobacterium hibernum RLF Bacillus mycoides PAK Sphingobacterium faecium CRC Bacteroidetes Bacillus mycoides JAC Sphingobacterium faecium VLVK Flavobacterium sp. nov. AMR • Minimize the risk of working with pathogenic organisms. Bacillus mycoides MB Chryseobacterium sp. nov CTM Sejongia sp. nov. JJC Bacillus mycoides MLH 30.5 Bacillus mycoides ROH Flavobacterium hibernum ECB Bacteroidetes • Integrate lab activities with associated lecture material. Bacillus mycoides CJB Flavobacterium hibernum KJA 30 25 20 15 10 5 0 Bacillus mycoides SMH Flavobacterium sp. nov AEC Nucleotide Substitutions (x100) Staphylococcus haemolyticus EMH 32.8 • DISCOVER NOVEL BACTERIAL SPECIES!!!! Staphylococcus epidermidis EFP Paenibacillus amylolyticus AKS 30 25 20 15 10 5 0 Figure 3. Phylogenetic tree resulting from Clustal W alignment of Arthrobacter nitroguaiacolicus ND Nucleotide Substitutions (x100) Serratia fonticola AP Actinobacteria 2007 unknown microbe 16S rRNA gene sequences. Serratia fonticola MW Serratia fonticola SHM Serratia fonticola BLR Figure 2. Phylogenetic tree resulting from Clustal W alignment of Student Learning Objectives: Serratia fonticola TL Serratia fonticola JW 2006 unknown microbe 16S rRNA gene sequences. Serratia fonticola LES • Phenotypic characterization of microbes, evaluation of Serratia fonticola ALM 2007 Lessons Learned Serratia fonticola CBS Serratia fonticola PST advantages & disadvantages. Serratia plymuthica JWT Serratia plymuthica NGV 2006 Lessons Learned Serratia plymuthica SLW • Universal rRNA Primers 27f & 1492r (Lane, • Molecular characterization of microbes, evaluation of Serratia plymuthica JJG Enterobacteria Serratia plymuthica RCS Serratia fonticola PV 1991) work as well as rRNA1 (356f) & rRNA2 advantages & disadvantages. Serratia grimesii ALR Serratia grimesii AR Serratia grimesii AW • Growth of initial sediment sample dilution (785r) (Newman, 2000) and yield the nearly full • Application of bioinformatic tools and databases to Yersinia intermedia TMB o o Yersinia intermedia CAB plates at 22 C or 37 C yield different types length rRNA gene PCR product. Yersinia intermedia AJL investigate evolutionary relationships among microbes. Yersinia intermedia KLK o Yersinia intermedia CC of organisms that can still grow at 30 C. Serratia fonticola JLG • Finding relevant information in the scientific literature. Serratia novum BDM • Bacteroidetes, -proteobacteria, Diversity Summary Pseudomonas libanensis CEC Pseudomonas libanensis PHS • Writing a primary literature-style research paper. Pseudomonas libanensis KEK Pseudomonads -proteobacteria & pseudomonadales Pseudomonas koreensis KMS isolates species genera families orders phyla Flexibacteriaceae GC o • The use of peer review in scientific publication Flexibacteriaceae KC are favored at 22 C. Flexibacteriaceae HTE Bacteroidetes 2004 47 15 4 4 3 2 38.4 35 30 25 20 15 10 5 0 2005 44 13 6 5 4 4 Nucleotide Substitutions (x100) • The NCBI databases contain many Figure 1. Phylogenetic tree resulting from Clustal W alignment of 2004+2005 91 23 8 7 5 4 2004 & 2005 unknown microbe 16S rRNA gene sequences. sequences for organisms whose names Methods (2007) have not been “officially” published. 2006 45 38 24 19 11 4 • Use Ribosomal Database Project (Cole et 2007 44 36 18 11 8 4 Creek sediment sample 2004-2005 Lessons Learned Colony Morphology 2006+2007 89 66 30 20 12 4 diluted, spread on TSA, al., 2007) to identify Type strain with incubated for 2d. Staining & Microscopy most similar sequence, then use BLAST2 2004-2007 180 79 33 21 12 4 • Students tend to choose the same types of (NCBI) to determine percent identity. Antibiotic Sensitivity colonies, resulting in poor diversity of Isolated colonies Compare to Bergey’s Manual unknown organisms. Instructor should select selected, restreaked to Carbohydrate Metabolism of Determinative Bacteriology obtain pure cultures. colonies from initial dilutions. Nitrogen/Amino Acid Metab. Outcomes References • Novel organisms attract students to research. . • Bergey’s Manual of Determinative Bacteriology • Bergey, D.H. Bergey's Manual of Determinative O2 & Temp. Requirements Suspend cells in dH2O, Develop hypothesis about does not allow one to distinguish among • 1 Independent study – Fall, 2005 Bacteriology. 9th ed. Baltimore: Williams & Wilkins, 1994. freeze thaw 2x identity of unknown organism Bacillus species. Avoid obvious Bacilli • 3 Research Methods Projects – Fall, 2006 Exoenzyme Production • Cole, J., Chai, B., Farris, R., Wang, Q., Kulam-Syed- • 1 Honors Project – Spring, 2007 Mohideen, A., McGarrell, D., Bandela, A., Cardenas, E., • High [salt] is worse than low [DNA] for • 3 Research Methods Projects – Fall, 2007 Garrity, G., and Tiedje, J. (2007).The ribosomal database Multiple Sequence Alignment Literature research, write lab PCR with universal • 1 ASM Poster #R-049 by Kellie Cicconi. project (RDP-II): introducting myRDP space and quality primers for 16S rRNA to construct phylogenetic tree report using journal format sequencing. Don’t concentrate DNA by drying. controlled public data. Nucleic Acids Research 35 gene (27f & 1492r) • Methods have been applied to other projects (database issue), D169-D172 • International Journal of Systematic and • Evaluation of biofilm community in a wastewater • Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic acid BLAST2 to determine percent Electronic submission, Evolutionary Microbiology (IJSEM) does not treatment sequencing batch reactor – ASM Poster techniques in bacterial systematics. E. Stackebrandt and M. identity (<97% = new species) anonymous peer review Gel purify PCR product, make free full text available until 2 years after #Q-079 by Brittane Miller Goodfellow, eds. New York, NY, John Wiley and Sons: 115- send to Geneway Res. • Bacterial Strain Collection has expanded dramatically. 175. for sequencing. publication! Ask library to purchase Compare to Type strains at Revise, submit lab report to subscription. • Raw materials for study in other courses - Fatty acid  Newman JD (2000) Molecular Phylogeny in the Ribosomal Database Project instructor for grading compositions are determined by Biochemistry class. Undergraduate Microbiology Laboratory. Focus on Microbiology Education 6(2):3-4.